ES2758051T3 - Humanized Universal Light Chain Mice - Google Patents

Abstract

A mouse whose genome comprises: (a) a humanized immunoglobulin heavy chain variable locus comprising at least one unreordered human VH segment, at least one unreordered human DH, and at least one unreordered human JH operably linked to a gene endogenous heavy chain constant region; (b) a humanized immunoglobulin light chain variable locus comprising not more than one, or not more than two, rearranged human light chain V / J sequences operably linked to an endogenous light chain constant region gene; and, (c) an ectopic nucleic acid sequence expressing a functional mouse ADAM6a protein or a functional ortholog or a functional homolog or a functional fragment thereof and an ectopic nucleic acid sequence expressing a functional mouse ADAM6b protein or a functional ortholog or functional homolog or a functional fragment thereof, the ADAM6 proteins, orthologs, homologs or fragments being functional in a male mouse, wherein said function is associated with male fertility, said mouse having the function of endogenous ADAM6 eliminated or reduced, and further characterized by its germline lacking a non-rearranged endogenous immunoglobulin Vκ gene segment and a non-rearranged endogenous immunoglobulin Jκ gene segment.

Description

DESCRIPTION

Humanized Universal Light Chain Mice

Field of the Invention

Genetically modified mice, cells, embryos, tissues and nucleic acids modified to produce antibodies and sequences encoding human immunoglobulin heavy chain variable domains, including bispecific antibodies and including bispecific antibodies comprising universal light chains. Compositions and methods involving genetically modified mice with germline replacements at the endogenous mouse heavy chain variable locus, comprising modified light chain loci expressing light chains derived from no more than one or two different light chain V gene segments , where the mice are also genetically modified in their germline in such a way that the male mice that carry these modifications are fertile. Genetically engineered mice expressing universal light chains and humanized heavy chain variable domains are provided, wherein the mice comprise ADAM6 activity that is functional in a male mouse.

Background

The development of antibodies for use as human therapeutic agents has a long and complex history. A significant advance has been the ability to essentially produce fully human antibody sequences for use in the design of effective human therapeutic agents with a reduced potential for immunogenicity. Today there are mice that are modified in their germ line to generate human antibody sequences derived from unrearranged genetic segments (heavy and light) as transgenes or as replacements at the endogenous mouse immunoglobulin loci. Replacing mouse variable sequences with human variable sequences at endogenous mice loci, as with humanized mice with VELOCIMMUNE®, allows the mouse immune system to function essentially normally. As a result, exposing these mice to an antigen of choice generates a wonderfully diverse and rich population of clonally selected B lymphocytes expressing high affinity somatically mutated human variable domains that can be used to produce fully human antibodies directed against the antigen of choice.

Human variable domains produced in humanized mice can be used to design fully human bispecific antibodies, i.e., binding proteins that are heavy chain heterodimers, where the identities and binding specificities of heavy chain variable domains differ. But selecting light chains that can effectively associate and express with heavy chain heterodimers does not have an easy solution. The development of human light chain variable domains for use in human therapeutic agents is certainly possible in humanized mice, but there are no easy solutions to select which light chains will effectively associate and express with heavy chains having desired binding characteristics, where light chains are not detrimental to the expression or binding behavior of both heavy chains.

Therefore, there remains a need in the art for compositions and methods for developing human immunoglobulin variable regions for use in human therapeutic agents, including human immunoglobulin variable regions generated from nucleic acid sequences at mouse immunoglobulin loci. endogenous.

Summary

Mice expressing human immunoglobulin variable domains are described which are suitable for use in bispecific binding proteins, including bispecific antibodies, wherein the mice comprise a humanization of an endogenous mouse heavy chain variable locus, wherein male mice that they comprise humanization they are fertile, and wherein the mice further comprise a humanization of an endogenous immunoglobulin light chain locus resulting in the mouse expressing a repertoire of immunoglobulin light chains derived from no more than one, or no more of two, VA and / or k gene segments.

Genetically engineered mice are selected which select suitable affinity matured human immunoglobulin heavy chain variable domains derived from a repertoire of unrearranged human heavy chain V, D, and J segments, wherein the human heavy chain variable domains matured by Affinity are associated and expressed with a humanized universal light chain. The humanized universal light chain is expressed from a locus comprising not more than one or not more than two human light chain V segments and a human J segment operably linked to a light chain constant gene, or not more than one or no more than two rearranged human nucleic acid sequences (VA / JA, Vk / Jk, VA / Jk, or Vk / JA) encoding a light chain variable domain operably linked to a light chain constant gene. In various embodiments, the humanized universal light chain domain is paired with a plurality of affinity matured human heavy chain variable domains, wherein the plurality of heavy chain variable domains they specifically bind to different epitopes or antigens. In particular, a mouse whose genome comprises:

(a) a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human Vh segment, at least one non-rearranged human Dh, and at least one non-rearranged human Jh operably linked to a heavy chain constant region gene endogenous;

(b) a humanized immunoglobulin light chain variable locus comprising not more than one, or not more than two, rearranged human light chain V / J sequences operably linked to an endogenous light chain constant region gene; and,

(c) an ectopic nucleic acid sequence expressing a functional mouse ADAM6a protein or a functional ortholog or a functional homolog or a functional fragment thereof and an ectopic nucleic acid sequence expressing a functional mouse ADAM6b protein or an ortholog functional or a functional homolog or a functional fragment thereof, the ADAM6 proteins, orthologs, homologs or fragments being functional in a male mouse, wherein said function is associated with male fertility,

said mouse having the function of eliminated or reduced endogenous ADAM6 and further characterized in that its germline lacks a non-rearranged endogenous immunoglobulin Vk gene segment and a non-rearranged endogenous immunoglobulin Jk gene segment.

Also provided is a mouse whose genome comprises:

a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human Vh segment, at least one non-rearranged human DH, and at least one non-rearranged human JH operably linked to an endogenous heavy chain constant region gene;

a humanized immunoglobulin light chain variable locus comprising a single rearranged human light chain V / J sequence operably linked to an endogenous light chain constant region gene, wherein the mouse expresses a single chimeric light chain, and wherein the mouse expresses from ectopic nucleic acid sequences a functional mouse ADAM6a protein and a functional mouse ADAM6b protein, whose ADAM6a and ADAM6b proteins are functional in a male mouse, where said function is associated with male fertility,

said mouse having the function of eliminated or reduced endogenous ADAM6 and further characterized in that its germline lacks a non-rearranged endogenous immunoglobulin Vk gene segment and a non-rearranged endogenous immunoglobulin Jk gene segment.

In addition, a mouse cell whose genome comprises:

a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human Vh segment, at least one non-rearranged human DH, and at least one non-rearranged human JH operably linked to an endogenous heavy chain constant region gene;

a humanized immunoglobulin light chain variable locus comprising not more than one, or not more than two, rearranged human light chain V / J sequences operably linked to an endogenous light chain constant region gene; and,

an ectopic nucleic acid sequence expressing a functional mouse ADAM6a protein or a functional ortholog or a functional homolog or a functional fragment thereof and an ectopic nucleic acid sequence expressing a functional mouse ADAM6b protein or a functional ortholog or a functional homolog or a functional fragment thereof, ADAM6 proteins, orthologs, homologs or fragments being functional in a male mouse, where said function is associated with male fertility,

said cell having the function of eliminated or reduced endogenous ADAM6 and further characterized in that it lacks a non-rearranged endogenous immunoglobulin gene segment Vk and a non-rearranged endogenous immunoglobulin gene Jk.

Nucleic acid constructs, cells, embryos, mice are also described, and methods are provided for producing mice comprising a humanized immunoglobulin heavy chain variable locus and a humanized immunoglobulin light chain variable locus, wherein the mouse expresses one of no more than two universal light chains and male mice show wild-type fertility.

Also described is a modified mouse comprising in its germ line a humanized immunoglobulin heavy chain variable locus in an endogenous mouse heavy chain locus and a humanized immunoglobulin light chain variable locus, where the mouse expresses a universal light chain , and wherein the mouse comprises a nucleic acid sequence encoding a mouse ADAM6 or an ortholog or a homolog or a functional fragment thereof. In several cases, the humanized immunoglobulin light chain variable locus is at an endogenous mouse light chain locus.

In one embodiment, the humanized immunoglobulin heavy chain variable locus comprises a replacement at the endogenous mouse heavy chain variable locus for all or substantially all functional mouse immunoglobulin heavy chain V, D and J gene segments with one or more human V, human D, and human J gene segments, wherein the one or more human V, D, and J segments are operably linked and capable rearranged to form a rearranged V / D / J gene that is operably linked to a heavy chain constant sequence.

In one embodiment, the mouse comprises a light chain locus that is engineered to produce a universal light chain, wherein the universal light chain is a light chain derived from a light chain locus comprising a single V / J sequence. rearranged light chain. Also disclosed is a mouse comprising an immunoglobulin light chain locus comprising a single human immunoglobulin light chain V segment that is capable of rearranging with a human light chain J gene segment (selected from one or a plurality of J segments ) and encoding a human light chain variable domain. In another case, the mouse comprises no more than two human V light chain segments at the light chain locus, each V segment of which is capable of rearranging itself with a human J gene segment (selected from one or a plurality of J segments. light chain) and encode a rearranged human light chain variable domain.

In one case, segment V of the single human light chain is operably linked to a segment of human light chain J selected from Jk1, Jk2, Jk3, Jk4 and Jk5, wherein the single segment of human light chain V is capable of rearranging itself. to form a sequence encoding a light chain variable region gene with any one or more human light chain J segments.

In one embodiment, the mouse comprises an endogenous light chain locus comprising a replacement of all or substantially all of the mouse V and J gene segments with no more than one, or no more than two, rearranged nucleic acid sequences (V / J). In one embodiment, the no more than one or no more than two rearranged nucleic acid sequences (V / J) are selected from Vk1-39Jk5, human Vk3-20Jk1, and a combination thereof.

In one embodiment, the mouse lacks a functional endogenous light chain locus that is capable of expressing a mouse light chain variable domain. In one embodiment, the mouse comprises a nucleic acid sequence encoding a variable domain of a universal light chain at a k locus. In one embodiment, the mouse comprises a nucleic acid sequence encoding a variable domain of a universal light chain at a locus A.

In one embodiment, the human rearranged V / J sequence is operably linked to a human or mouse leader sequence. In one embodiment, the leader sequence is a mouse leader sequence. In a specific embodiment, the mouse leader sequence is a mouse Vk3-7 leader sequence.

In one embodiment, the rearranged V / J sequence is operably linked to an immunoglobulin promoter sequence. In one embodiment, the promoter sequence is a human promoter sequence. In a specific embodiment, the human immunoglobulin promoter is a human Vk3-15 promoter.

In one embodiment, the rearranged (V / J) sequence is operably linked to a mouse CK gene.

In one embodiment, the rearranged (V / J) sequence is present at a k light chain locus, and the k light chain locus comprises a mouse k intronic enhancer, a mouse 3'k enhancer, or both an intronic and a 3 'enhancer. In a specific embodiment, the k locus is an endogenous k locus.

In one embodiment, the mouse comprises a k locus comprising a sequence encoding a universal light chain variable domain, and the mouse comprises a non-functional immunoglobulin lambda (A) light chain locus. In a specific embodiment, the A light chain locus comprises a deletion of one or more locus sequences, where the one or more deletions render the A light chain locus unable to rearrange itself to form a light chain gene. . In another embodiment, all or substantially all V gene segments of the light chain A locus are deleted. In another embodiment, the mouse comprises a deletion of all or substantially all of the endogenous light chain variable loci.

In one embodiment, the mouse further comprises in its germline a sequence selected from a 5 'mouse intronic k enhancer with respect to the rearranged immunoglobulin light chain sequence or the unrearranged genetic segments, a mouse 3'k enhancer, and a combination of them.

In one embodiment, the mouse universal light chain variable domain sequence comprises one or more somatic hypermutations; In one embodiment, the variable domain sequence comprises a plurality of somatic hypermutations.

In one embodiment, the mouse produces a universal light chain comprising a somatically mutated human variable domain. In one embodiment, the light chain comprises a somatically mutated human variable domain derived from a human V segment, a human J segment, and a mouse Ck gene. In one embodiment, the mouse does not express an A light chain.

In one embodiment, the human variable sequence is a rearranged human Vk1-39Jk5 sequence and the mouse expresses a reverse chimeric light chain comprising (i) a variable domain derived from Vk1-39Jk5 and (ii) a mouse C ^l ; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse C ^h and (ii) a somatically mutated human heavy chain variable domain. In one embodiment, the mouse expresses a light chain that is somatically mutated. In one embodiment, the C ^l is a mouse Ck.

In one embodiment, the human variable sequence is a human sequence reordered Vk3-20Jk1 and mouse expresses a reverse chimeric light chain comprising (i) a variable domain Vk3-20Jk1 derivative, (ii) a C ^l mouse; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse C ^h and (ii) a somatically mutated human heavy chain variable domain.

In one embodiment, the mouse comprises both a rearranged human Vk1-39Jk5 sequence and a rearranged human Vk3-20Jk1 sequence, and the mouse expresses a reverse chimeric light chain comprising (i) a light chain comprising a variable domain derived from the sequence Vk1-39Jk5 or the Vk3-20Jk1 sequence and (ii) a mouse C ^l ; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse C ^h and (ii) a somatically mutated human heavy chain variable domain. In one embodiment, the mouse expresses a light chain that is somatically mutated. In one embodiment, the C ^l is a mouse Ck.

In one embodiment, the mouse expresses a reverse chimeric antibody comprising a light chain comprising a mouse Ck and a somatically mutated human variable domain derived from a rearranged human Vk1-39Jk5 sequence or a rearranged human Vk3-20Jk1 sequence, and a chain Heavy comprising a mouse C ^h and a somatically mutated human heavy chain variable domain, wherein the mouse does not express a fully mouse antibody and does not express a fully human antibody. In one embodiment, the mouse comprises a k light chain locus comprising a replacement of endogenous mouse k light chain gene segments with the rearranged human Vk1-39Jk5 sequence or the rearranged human Vk3-20Jk1 sequence, and comprises a replacement for the all or substantially all endogenous mouse heavy chain V, D and J gene segments with a complete or substantially complete repertoire of human heavy chain V, D and J gene segments.

In one aspect, there is provided a genetically modified mouse expressing a single k light chain derived from no more than one, or no more than two, rearranged k light chain sequences, wherein the mouse, upon immunization with antigen, displays a serum titer that is comparable to a wild type mouse immunized with the same antigen. In a specific embodiment, the mouse expresses a single k light chain sequence, wherein the single k light chain sequence is derived from no more than one rearranged k light chain sequence. In one embodiment, the serum titer is characterized as total immunoglobulin. In a specific embodiment, the serum titer is characterized as IgM specific titer. In a specific embodiment, the serum titer is characterized as IgG specific titer. In a more specific embodiment, the rearranged k light chain sequence is selected from a sequence Vk1-39Jk5 and Vk3-20Jk1. In one embodiment, the rearranged k light chain sequence is a Vk1-39Jk5 sequence. In one embodiment, the rearranged k light chain sequence is a Vk3-20Jk1 sequence.

In one aspect, the provided genetically modified mouse expresses a plurality of immunoglobulin heavy chains associated with a single light chain sequence. The heavy chain comprises a human sequence. The human sequence includes at least one variable sequence and can also comprise a human sequence selected from a CH1, a hinge, a CH2, a CH3, and a combination thereof. The single light chain comprises a human sequence. The human sequence includes at least one variable sequence and can also comprise a human sequence selected from a constant sequence and a combination thereof. In one embodiment, the mouse comprises a deactivated endogenous immunoglobulin locus and expresses the heavy chain of an extrachromosomal transgene or episome. In one embodiment, the mouse comprises a replacement at an endogenous mouse locus of some or all of the endogenous mouse heavy chain gene segments (i.e., V, D, J), and / or some or all of the chain constant sequences endogenous mouse weighing (eg, CHi, hinge, CH2, CH3, or a combination thereof), and / or some or all of the endogenous mouse light chain sequences (eg, V, J, constant, or a combination thereof), with one or more human immunoglobulin sequences.

In one embodiment, the mouse, after rearrangement of one or more V, D and J gene segments, the mouse comprises in its genome at least one nucleic acid sequence encoding a mouse ADAM6 gene or a homolog or ortholog or a functional fragment of it. In one embodiment, after rearrangement, the mouse comprises in its genome at least two nucleic acid sequences encoding a mouse ADAM6 gene or a homolog or ortholog or a functional fragment thereof. In one embodiment, after rearrangement, the mouse comprises in its genome at least one nucleic acid sequence encoding a mouse ADAM6 gene or a homolog or ortholog or a functional fragment thereof. In one embodiment, the mouse comprises the ADAM6 gene or a homolog or an ortholog or a functional fragment thereof in a B lymphocyte.

[Removed].

Male mice comprising an ADAM6 sequence or a homolog or ortholog or a functional fragment thereof are also disclosed at a location in the mouse genome that approximates the location of the allele. endogenous mouse ADAM6, eg, 3 'from a sequence of the final V gene segment and 5' from an initial D gene segment.

In one embodiment, male mice comprise an ADAM6 sequence or a homolog or an ortholog or a functional fragment thereof flanked upstream, downstream, or upstream and downstream (with respect to the transcription direction of the ADAM6 sequence) of a nucleic acid sequence encoding an immunoglobulin variable region gene segment. In a specific embodiment, the gene segment of the immunoglobulin variable region is a human gene segment. In one embodiment, the immunoglobulin variable region gene segment is a human gene segment, and the sequence encoding mouse ADAM6 or an ortholog or homolog or a functional fragment thereof in a mouse is between human V gene segments; In one embodiment, the mouse comprises two or more human V gene segments, and the sequence is in a position between the final V gene segment and the penultimate V gene segment; in one embodiment, the sequence is in a position that follows the final V gene segment and the first D gene segment. In the embodiments described herein, reference to "ADAM6" means "ADAM6 and ADAM6b".

In one embodiment, the humanized immunoglobulin heavy chain variable locus lacks an endogenous mouse ADAM6 gene. In one embodiment, the humanized immunoglobulin heavy chain variable locus comprises an ADAM6 gene that is functional in a male mouse. In a specific embodiment, the ADAM6 gene that is functional in the male mouse is a mouse ADAM6 gene, and the mouse ADAM6 gene is placed within or immediately adjacent to the humanized immunoglobulin heavy chain variable locus.

In one embodiment, the humanized immunoglobulin heavy chain variable locus lacks an endogenous mouse ADAM6 gene, and the mouse comprises an ectopic ADAM6 sequence that is functional in a male mouse. In one embodiment, the ectopic ADAM6 gene that is functional in the male mouse is a mouse ADAM6 gene. In one embodiment, the mouse ADAM6 gene is on the same chromosome as the humanized immunoglobulin heavy chain variable locus. In one embodiment, the mouse ADAM6 gene is on a different chromosome than the humanized immunoglobulin heavy chain variable locus. In one embodiment, the mouse ADAM6 gene is at an episome.

In one case, the mouse comprises a first endogenous heavy chain allele and a second endogenous heavy chain allele, and the first endogenous heavy chain allele comprises a deletion of a mouse ADAM6 locus, and the first endogenous heavy chain allele comprises a replacement of all or substantially all functional mouse V, D, and J segments with one or more human V, D, and J segments. In one embodiment, the first and second endogenous heavy chain alleles each comprise a deletion of an endogenous mouse ADAM6 locus, and the first and second endogenous heavy chain alleles comprise a replacement for all or substantially all V segments. Mouse, D and J functional with one or more human V, D and J segments. In one embodiment, the first and / or second allele comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 or an ortholog or homolog or a functional fragment thereof. In one embodiment, the ectopic nucleic acid sequence is located 3 '(with respect to the transcriptional directionality of the variable heavy chain locus) of a final mouse V gene segment and is located 5' (with respect to the transcriptional directionality of the constant sequence) of a mouse (or human / mouse chimeric) heavy chain constant gene or fragment thereof (eg, a nucleic acid sequence encoding C ^h 1 and / or hinge C ^h 2 and / or C ^h 3 human and / or mouse). In one embodiment, the ectopic nucleic acid sequence is located downstream (with respect to the transcription direction of the V segment locus) of a V segment and upstream of a D segment. In one embodiment, the ectopic nucleic acid sequence It is located between the penultimate segment V plus 3 'and the last segment V plus 3'. In a specific embodiment, the ectopic nucleic acid sequence is located between the human V segment V ^h 1-2 and the human V segment V ^h 6-1. In one embodiment, the nucleotide sequence between the two human V gene segments is placed in opposite transcription orientation with respect to the human V gene segments. In a specific embodiment, the nucleotide sequence encodes 5 'to 3' with respect to the transcription direction of the ADAM6 genes and the ADAM6a sequence followed by an ADAM6b sequence. In a specific embodiment, the ADAM6 genes are oriented in opposite transcriptional orientation compared to the upstream and downstream flanking V segments.

A nucleic acid sequence encoding mouse ADAM6a or a functional fragment thereof and / or a sequence encoding mouse ADAM6b or a functional fragment thereof is described, wherein ADAM6a and / or ADAM6b or one or more fragments Functional thereof are operatively linked to a promoter. The promoter can be a human promoter. The promoter may be the mouse ADAM6 promoter. The ADAM6 promoter may comprise a sequence located between the first codon of the first ADAM6 gene closest to the mouse D ^h plus 5 'gene segment and the recombination signal sequence of the D ^h plus 5' gene segment, where 5 'is indicated. with respect to transcription direction of the mouse immunoglobulin genes. The promoter can be a viral promoter. The viral promoter can be a cytomegalovirus (CMV) promoter. The promoter can be a ubiquitin promoter.

In one embodiment, the mouse ADAM6a and / or ADAM6b are selected from ADAM6a of SEQ ID NO: 1 and / or sequence ADAM6b of SEQ ID NO: 2. In one embodiment, the mouse ADAM6 promoter is a promoter of s Eq ID NO: 3. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3 directly upstream (with respect to the ADAM6a transcription direction) of the first codon of ADAM6a and extends to the end of SEQ ID NO: 3 upstream of the coding region of ADAM6. In other specific embodiment, the ADAM6 promoter is a fragment that extends from about 5 to about 20 nucleotides upstream of the ADAM6a start codon to about 0.5 kb, 1kb, 2 kb or 3 kb or more upstream of the start codon of ADAM6a.

In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 3 or a fragment thereof as when placed in a mouse that is infertile or has low fertility due to lack of ADAM6, improves fertility, or restores fertility to a wild type fertility. In one embodiment, SEQ ID NO: 3 or a fragment thereof confers on a male mouse the ability to produce a sperm that is capable of passing through a female mouse oviduct to fertilize a mouse egg.

The mice comprise a nucleic acid sequence encoding an ADAM6 protein, or an ortholog or a homolog or a fragment thereof, that is functional in a male mouse. In a specific embodiment, the nucleic acid sequence is within or adjacent to a human nucleic acid sequence that comprises one or more immunoglobulin variable region gene segments. In one embodiment, the one or more immunoglobulin variable region gene segments is at a modified endogenous mouse immunoglobulin heavy chain variable locus. In one embodiment, the modification comprises replacing all or substantially all functional mouse immunoglobulin heavy chain variable gene segments with a plurality of unrearranged human heavy chain gene segment segments that are operably linked to a region gene endogenous mouse constant. In a specific embodiment, the nucleic acid sequence is between two human V segments. In a specific embodiment, the nucleic acid sequence is between a human V segment and a human D segment. In a specific embodiment, the nucleic acid sequence is between a human D segment and a human J segment. In a specific embodiment, the nucleic acid sequence is upstream of the human V segment plus 5 '(with respect to the transcription direction of segment V). In a specific embodiment, the nucleic acid sequence is between a human J segment and an endogenous mouse heavy chain constant region gene sequence.

In one embodiment, male mice are capable of generating offspring by mating, with a frequency that is almost the same as that of a wild type mouse. In one embodiment, male mice produce sperm that can travel from a mouse's uterus through a mouse oviduct to fertilize a mouse egg; In a specific embodiment, the sperm from the mice transits through the oviduct almost as efficiently as the sperm from a wild type mouse. In one embodiment, approximately 50% or more of the sperm produced in the mouse show the ability to enter and / or transit an oviduct to fertilize a mouse egg.

The mouse lacks a functional endogenous ADAM6 locus, where the mouse comprises an ectopic nucleotide sequence that complements the loss of mouse ADAM6 function in a male mouse. In one embodiment, the ectopic nucleotide sequence confers on the male mouse the ability to produce offspring that is comparable to a corresponding wild-type male mouse containing a functional endogenous ADAM6 gene. In one embodiment, the sequence confers on the mouse the ability to form a complex of ADAM2 and / or ADAM3 and / or ADAM6 on the surface of the mouse sperm. In one embodiment, the sequence confers on the mouse the ability to travel from a mouse uterus through a mouse oviduct to a mouse egg to fertilize the egg.

In one embodiment, the mouse lacks a functional endogenous ADAM6 locus and comprises an ectopic nucleotide sequence encoding an ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse and where the male mouse produces at least about 50%, 60%, 70%, 80%, or 90% of the number of litters produced by a wild-type mouse of the same age and strain over a period of six months.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces at least about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, about 6 times, about 7 times, about 8 times, or about 10 times or more of progeny when breeding for a period of six months as a mouse of the same age and the same or similar strain lacking the functional endogenous ADAM6 gene and lacking of the ectopic nucleotide sequence that reproduces for substantially the same period of time and under substantially the same conditions.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces an average of at least about 2 times, 3 times or 4 times more puppies per litter in a 4 or 4 breeding period 6 months that a mouse that lacks the functional endogenous ADAM6 gene and that lacks the ectopic nucleotide sequence, and that reproduces during the same period of time.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence is a male mouse, and the male mouse produces sperm that when recovered from the oviducts approximately 5-6 hours after copulation reflects a migration in the oviduct that is at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times , 100 times, 110 times, or 120 times or more than the sperm from a mouse lacking the functional endogenous ADAM6 gene and lacking the ectopic nucleotide sequence.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence when copulated with a female mouse generates sperm that is capable of traversing the uterus and entering and traversing the oviduct within approximately 6 hours with efficacy which is roughly equal to that of sperm from a wild-type mouse.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces approximately 1.5 times, approximately 2 times, approximately 3 times, or approximately 4 times or more litters in a comparable time period than a mouse lacking the functional ADAM6 gene and lacking the ectopic nucleotide sequence.

Also described is a mouse comprising a humanized endogenous mouse immunoglobulin heavy chain variable locus and a modification of a mouse immunoglobulin light chain locus, wherein the mouse expresses a B lymphocyte comprising an immunoglobulin heavy chain sequence. rearranged human operably linked to a human or mouse heavy chain constant region gene sequence, and the B lymphocyte comprises in its genome (eg, a B lymphocyte chromosome) a gene encoding an ADAM6 or ortholog or a homolog or fragment thereof that is functional in a male mouse (eg, a mouse ADAM6 gene, eg, mouse ADAM6a and / or mouse ADAM6b), wherein the variable domains of immunoglobulin light chains A ok mice derive from no more than one or no more than two light chain V gene segments.

In one case, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human heavy chain V, D and / or J sequence; a mouse heavy chain V, D and / or J sequence; a human and mouse light chain V and / or J sequence. In one case, the heavy chain constant region gene sequence comprises a human or mouse heavy chain sequence selected from the group consisting of a C ^h 1, a hinge, a C ^h 2, a C ^h 3 and a combination thereof.

In one aspect, the provided mouse is suitable for producing antibodies having the same light chain, where all or substantially all of the antibodies produced in the mouse are expressed with the same light chain, where the light chain comprises a human variable domain , and wherein the antibodies comprise a heavy chain comprising a human variable domain.

In one aspect, the provided mouse is characterized by the inability of the mouse to produce an B cell expressing an immunoglobulin light chain variable domain that is derived from a rearranged light chain sequence that is not a human Vk1-39Jk5 sequence or sequence Vk3-20Jk1 human.

In one embodiment, the mouse displays a k: A light chain ratio that is approximately the same as that of a mouse comprising a wild type complement of immunoglobulin light chain V and J gene segments.

In one aspect, there is provided a mouse as described herein that expresses an immunoglobulin light chain derived from a human Vk1-39Jk5 sequence or a human Vk3-20Jk1 sequence, wherein the mouse comprises a replacement for all or substantially all endogenous mouse heavy chain variable region gene segments with one or more human heavy chain variable region gene segments, and the mouse displays a proportion of (a) CD19 + B lymphocytes expressing an immunoglobulin having one chain light A, a (b) CD19 + B lymphocytes expressing an immunoglobulin having a light chain k, from about 1 to about 20.

In one embodiment, the mouse expresses a single k light chain, wherein the single k light chain is derived from a human Vk1-39Jk5 sequence and the ratio of CD19 + B lymphocytes expressing an immunoglobulin having an A light chain to CD19 + B lymphocytes that they express an immunoglobulin having a light chain k is from about 1 to about 20; in one embodiment, the ratio is from about 1 to at least about 66; in a specific embodiment, the ratio is from about 1 to 66.

In one embodiment, the mouse expresses a single k light chain, wherein the single k light chain is derived from a human Vk3-20Jk5 sequence and the ratio of CD19 + B lymphocytes expressing an immunoglobulin having an A light chain to CD19 + B lymphocytes that they express an immunoglobulin having a light chain k is from about 1 to about 20; in one embodiment, the ratio is from about 1 to about 21. In specific embodiments, the ratio is from 1 to 20 or 1 to 21.

In one embodiment, the percentage of IgK + IgA + B lymphocytes in the mouse is approximately the same as in a wild type mouse. In a specific embodiment, the percentage of IgK + IgA + B lymphocytes in the mouse is about 2 to about 6 percent. In a specific embodiment, the percentage of IgK + IgA + B lymphocytes in a mouse where the single rearranged k light chain is derived from a Vk1-39Jk5 sequence is from about 2 to about 3; in a specific embodiment, approximately 2.6. In a specific embodiment, the percentage of IgK + IgA + B lymphocytes in a mouse where the single rearranged k light chain is derived from a Vk3-20Jk1 sequence is from about 4 to about 8; in a specific embodiment, approximately 6.

In one embodiment, the mouse does not comprise a modification that reduces or eliminates the mouse's ability to somatically mutate any mouse light chain functional locus. In one embodiment, the single light chain functional locus in the mouse expresses a light chain comprising a human variable domain derived from a rearranged sequence selected from a human Vk1-39Jk5 sequence, a human Vk3-20Jk1 sequence, and a combination thereof. .

In one aspect, we provide the genetically modified mouse that expresses a single k light chain derived from no more than one, or no more than two, rearranged k light chain sequences, wherein the mouse demonstrates the use of the k light chain that is approximately 100 times or more, at least approximately 200 times or more, at least approximately 300 times or more, at least approximately 400 times or more, at least approximately 500 times or more, at least approximately 600 times or more, at least approximately 700 times or more, at least about 800 times or more, at least about 900 times or more, at least about 1000 times or more, greater than using the same light chain k (i.e. derived from the same segment V and the same J segment, or derived from the same rearranged V / J segment) displayed by a mouse supporting a complete or substantially complete human k-light chain locus. In a specific embodiment, the mouse carrying a complete or substantially complete human k light chain locus lacks a functional, unordered mouse k light chain sequence. In a specific embodiment, the mouse expresses the unique k light chain of no more than one rearranged k light chain sequence. In one embodiment, the mouse comprises a copy of a rearranged k light chain sequence (eg, a heterozygote). In one embodiment, the mouse comprises two copies of a rearranged k light chain sequence (eg, a homozygote). In a more specific embodiment, the rearranged k light chain sequence is selected from a sequence Vk1-39Jk5 and Vk3-20Jk1. In one embodiment, the rearranged k light chain sequence is a Vk1-39Jk5 sequence. In one embodiment, the rearranged k light chain sequence is a Vk3-20Jk1 sequence.

In one aspect, the genetically modified mouse provided expresses a single light chain derived from no more than one, or no more than two, rearranged k light chain sequences, wherein the light chain in the genetically modified mouse exhibits a level of expression that It is at least 10 times to approximately 1,000 times, 100 times to approximately 1,000 times, 200 times to approximately 1,000 times, 300 times to approximately 1,000 times, 400 times to approximately 1,000 times, 500 times to approximately 1,000 times, 600 times to approximately 1,000. times, 700 times to about 1,000 times, 800 times to about 1,000 times, or 900 times to about 1,000 times greater than the expression of the same rearranged light chain shown by a mouse carrying a complete or substantially human k-light chain variable locus full. In one embodiment, the light chain comprises a human sequence. In one embodiment, the single light chain is derived from a rearranged k light chain sequence selected from a human Vk1-39Jk5, a human Vk3-20Jk1, and a combination thereof.

In one embodiment, the level of light chain expression, in order to compare light chain expression with expression in a mouse comprising a substantially fully humanized light chain variable locus, is characterized by quantitating mRNA from the sequence of the transcribed light chain (of the one or two rearranged sequences), and comparing them to the transcribed light chain sequence of a mouse carrying a complete or substantially complete light chain locus.

Also disclosed is a method of producing an antibody, comprising expressing in a cell (a) a first human heavy chain variable domain nucleic acid sequence from an immunized mouse as described herein fused to a C gene sequence human ^h ; (b) a human light chain variable domain nucleic acid sequence from an immunized mouse as described herein fused with a sequence of the human C ^l gene; and, (c) maintaining the cell under conditions sufficient to express a fully human antibody and isolate the antibody. In one embodiment, the cell comprises a second human heavy chain variable domain nucleic acid sequence from a second immunized mouse as described herein fused with a human C ^h gene sequence, the first chain nucleic acid sequence heavy encodes a first heavy chain variable domain that recognizes a first epitope, and the second heavy chain nucleic acid sequence encodes a second heavy chain variable domain that recognizes a second epitope, where the first epitope and the second epitope are not identical.

Also disclosed is a method of producing an epitope-binding protein, which comprises exposing a mouse as described herein with an antigen comprising an epitope of interest, maintaining the mouse under conditions sufficient for the mouse to generate a molecule of immunoglobulin that specifically binds to the epitope of interest and isolating the immunoglobulin molecule that specifically binds to the epitope of interest; wherein the epitope binding protein comprises a heavy chain comprising a somatically mutated human variable domain and a mouse C ^h , associated with a light chain comprising a mouse C ^l and a human variable domain derived from a Vk1-39Jk5 rearranged human or a rearranged human Vk3-20Jk1.

Also disclosed is a method of producing a bispecific antigen binding protein, comprising exposing a first mouse as described herein to a first antigen of interest comprising a first epitope, exposing a second mouse as described herein. document a second antigen of interest comprising a second epitope, allow the first and second mice to mount immune responses to the antigens of interest, identify in the first mouse a first human heavy chain variable region that binds to the first epitope of the first antigen of interest, identify in the second mouse a second human heavy chain variable region that binds to the second epitope of the second antigen of interest, produce a first fully human heavy chain gene encoding a first heavy chain that binds to the first epitope of the first antigen of interest, producing a fully human second heavy chain gene encoding a a second heavy chain that binds to the second epitope of the second antigen of interest, expressing the first heavy chain and the second heavy chain in a cell expressing a single fully human light chain derived from a human Vk1-39 or Vk3-20 gene segment human to form a bispecific antigen-binding protein, and isolate the bispecific antigen-binding protein.

In one case, the first antigen and the second antigen are not identical.

In one case, the first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical. In one case, the binding of the first heavy chain variable region to the first epitope does not block the binding of the second heavy chain variable region to the second epitope.

In one case, the first antigen is selected from a soluble antigen and a cell surface antigen (eg, a tumor antigen), and the second antigen comprises a cell surface receptor. In a specific embodiment, the cell surface receptor is an immunoglobulin receptor. In a specific embodiment, the immunoglobulin receptor is an Fc receptor. In one case, the first antigen and the second antigen are the same cell surface receptor, and the binding of the first heavy chain to the first epitope does not block the binding of the second heavy chain to the second epitope.

In one embodiment, the light chain variable domain of the light chain comprises from 2 to 5 somatic mutations. In one embodiment, the light chain variable domain is a somatically mutated cognate light chain expressed in a B lymphocyte from the first or second mouse immunized with the first or second heavy chain variable domain.

In one aspect, a cell is provided that expresses an epitope-binding protein, wherein the cell comprises: (a) a human nucleotide sequence encoding a human light chain variable domain derived from a rearranged human Vk1-39Jk5 or a rearranged human Vk3-20Jk1, where the human nucleic acid sequence is fused (directly or through a linker) to a human immunoglobulin light chain constant domain nucleic acid sequence (eg, a domain DNA sequence human k constant); and, (b) a first human heavy chain variable domain nucleic acid sequence encoding a human heavy chain variable domain derived from a first human heavy chain variable domain nucleotide sequence, wherein the first nucleotide sequence of Human heavy chain variable domain is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain nucleic acid sequence (eg, a sequence of human IgG1, IgG2, IgG3, IgG4 or IgE); wherein the epitope binding protein recognizes a first epitope. In one embodiment, the epitope-binding protein binds to the first epitope with a dissociation constant of less than 10-6M, less than 10-8M, less than 10-9M, less than 10-10M, less than 10-11 M, or less than 10-12 M. In one embodiment, the cell comprises a second human nucleotide sequence encoding a second human heavy chain variable domain, where the second human sequence is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain nucleic acid sequence, and wherein the second human heavy chain variable domain does not specifically recognize the first epitope (eg, exhibits a dissociation constant of, for example, 10-6 M, 10-5 M, 10-4 M, or higher), and wherein the epitope-binding protein binds to both the first epitope and the second epitope, and wherein the first and second heavy chains of immunoglobulin are each associated with a light chain according or with (a). In one embodiment, the second domain V ^h joins the second epitope with a dissociation constant that is less than 10-6 M, less than 10-7 M, less than 10-8M, less than 10-9 M, less at 10-10 M, less than 10-11 M or less than 10-12 M.

In one embodiment, the epitope-binding protein comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, each associated with a universal light chain (eg, a light chain derived from a rearranged human light chain variable sequence. selected from a human Vk1-39Jk5 or a human Vk3-20Jk1), wherein the first immunoglobulin heavy chain binds to a first epitope with a dissociation constant in the nanomolar range (eg, 1 nM to 100 nM) to picomolar (eg, 1 pM to 100 pM), the second immunoglobulin heavy chain binds a second epitope with a dissociation constant in the nanomolar to picomolar range (eg, 1 pM at 100 nM), the first epitope, and the second epitope are not identical, the first immunoglobulin heavy chain does not bind to the second epitope or bind to the second epitope with a dissociation constant weaker than the micromolar interval (for example, the interv allo millimolar), the second immunoglobulin heavy chain does not bind to the first epitope or bind to the first epitope with a dissociation constant weaker than the micromolar interval (eg, the millimolar interval) and one or more of the variable domains (i.e. one or more of the light chain variable domain, the heavy chain variable domain of the first immunoglobulin heavy chain, and the heavy chain variable domain) of the second immunoglobulin heavy chain is somatically mutated. In one embodiment, binding of the epitope binding protein to the first epitope does not block the binding of the epitope binding protein to the second epitope.

In one embodiment, the first immunoglobulin heavy chain comprises a wild-type protein A binding determinant, and the second heavy chain lacks a wild-type protein A binding determinant. In one embodiment, the first immunoglobulin heavy chain binds to protein A under isolation conditions, and the second immunoglobulin heavy chain does not bind protein A or bind protein A at least 10 times, about one hundred times. or a thousand times weaker than the first immunoglobulin heavy chain binds to protein A under isolated conditions. In a specific embodiment, the first and second heavy chains are IgG1 isotypes, where the second heavy chain comprises a modification selected from 95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein the first heavy chain lacks such modification.

In one aspect, a mouse, embryo, or cell as described herein comprises a k light chain locus that retains endogenous regulatory or control elements, eg, a mouse k intronic enhancer, a mouse 3'k enhancer , or both an intronic enhancer and a 3 'enhancer, where regulatory or control elements facilitate somatic mutation and affinity maturation of an expressed sequence of the k light chain locus.

In one aspect, there is provided a mouse cell that is isolated from a mouse as described herein. Therefore, a mouse cell whose genome comprises: is also provided.

a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human V ^h segment, at least one non-rearranged human D ^h , and at least one non-rearranged human J ^h operably linked to a heavy chain constant region gene endogenous;

a humanized immunoglobulin light chain variable locus comprising not more than one, or not more than two, rearranged human light chain V / J sequences operably linked to an endogenous light chain constant region gene; and,

an ectopic nucleic acid sequence expressing a functional mouse ADAM6a protein or a functional ortholog or a functional homolog or a functional fragment thereof and an ectopic nucleic acid sequence expressing a functional mouse ADAM6b protein or a functional ortholog or a functional homolog or a functional fragment thereof, ADAM6 proteins, orthologs, homologs or fragments being functional in a male mouse, where said function is associated with male fertility,

said cell having the function of eliminated or reduced endogenous ADAM6 and further characterized in that it lacks a non-rearranged endogenous immunoglobulin Vk gene segment and a non-rearranged endogenous immunoglobulin Jk gene segment.

In one embodiment, the cell is an ES cell. In one embodiment, the cell is a lymphocyte. In one embodiment, the lymphocyte is a B lymphocyte. In one embodiment, B lymphocyte expresses a chimeric heavy chain comprising a variable domain derived from a human V gene segment; and a light chain derived from (a) a rearranged human Vk1-39 / J sequence, (b) a rearranged human Vk3-20 / J sequence, or (c) a combination thereof; wherein the heavy chain variable domain is fused to a mouse constant region and the light chain variable domain is fused to a mouse or human constant region. In one embodiment, the cell is a B lymphocyte and the B lymphocyte comprises a sequence encoding a rearranged human immunoglobulin heavy chain variable domain and a sequence encoding a universal light chain variable domain, wherein the B lymphocyte comprises in a chromosome a nucleic acid sequence that encodes an ADAM6 protein or an ortholog or a homolog or a fragment thereof that is functional in a male mouse; In one embodiment, the mouse B lymphocyte comprises two alleles of the nucleic acid sequence.

In one aspect, the provided mouse cell comprises a first chromosome comprising a humanized immunoglobulin heavy chain locus comprising unrearranged human V, D, and J segments; a second chromosome comprising a humanized immunoglobulin light chain locus that encodes or is capable of rearranging to encode a light chain, wherein the light chain locus comprises no more than one or no more than two light chain V / J sequences rearrangements operably linked to a constant light chain gene; and a third chromosome comprising a nucleic acid sequence encoding a mouse ADAM6 or an ortholog or a homolog or a fragment thereof that is functional in a male mouse. In one embodiment, the first and third chromosomes are the same. In one embodiment, the second and third chromosomes are the same. In one embodiment, the first, second, and third chromosomes are each different. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 or an ortholog or homolog or a functional fragment thereof is present in two copies. In one embodiment, the cell is a somatic cell. In a specific embodiment, the somatic cell is a B lymphocyte. In one embodiment, the cell is a germ cell.

In one aspect, a hybridoma is provided, wherein the hybridoma is produced with mouse B lymphocyte as described herein. In a specific embodiment, the B lymphocyte is from a mouse as described herein that has been immunized with an antigen comprising an epitope of interest, and the B lymphocyte expresses a binding protein that binds to the epitope of interest, the binding protein has a somatically mutated human heavy chain variable domain and a mouse heavy chain constant region, and has a Vk1-derived human light chain variable domain Rearranged human -39Jk5 or a rearranged human V k3-20Jk1 and a mouse C ^l .

Also disclosed is a cell comprising a fully human heavy chain gene comprising a nucleic acid sequence encoding a mouse first heavy chain variable domain as described herein, and a fully human light chain gene that it comprises a nucleic acid sequence encoding a universal light chain sequence as described herein. In one case, the cell further comprises a nucleic acid sequence encoding a second heavy chain variable domain of a mouse as described herein, wherein the first and second heavy chain variable domain are different. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell that expresses a viral nucleic acid sequence (eg, a PERC.6 ™ cell).

Also described is a mouse embryo, wherein the embryo comprises a donor ES cell derived from a mouse as described herein.

Also described is the use of a mouse embryo comprising a genetic modification as described herein, wherein the use comprises producing a genetically modified mouse as described herein.

Also described is a human heavy chain variable domain and an amino acid sequence of the human light chain variable domain of an antibody produced in a mouse as described herein.

Also disclosed is a human heavy chain variable domain nucleotide sequence and a human light chain variable domain nucleotide sequence of an antibody produced in a mouse as described herein.

Also disclosed is an antigen binding antibody or protein or an antigen binding fragment thereof (eg, Fab, F (ab) 2, scFv) produced in a mouse as described herein.

In one aspect, the provided mouse is produced using a targeting vector, a nucleotide construct, or a cell as described herein.

In one aspect, the mouse provided a mating progeny of a first mouse as described herein with a second mouse that is a wild type or genetically modified mouse.

Also described is the use of a mouse as described herein to produce a fully human antibody, or a fully human antigen binding protein comprising an immunoglobulin variable domain or a functional fragment thereof.

Also described is the use of a mouse or tissue or cell as described herein to produce a fully human bispecific antibody.

Also described is the use of a nucleic acid sequence produced by a mouse as described herein, wherein the use comprises expressing the nucleic acid sequence in the production of a human therapeutic agent.

Also described is the use of a mouse as described herein to produce an immortalized cell line.

Also described is the use of a mouse as described herein to produce a hybridoma or quadroma.

Also described is the use of a mouse as described herein to produce a nucleic acid sequence encoding an immunoglobulin variable region or a fragment thereof is provided. In one embodiment, the nucleic acid sequence is used to produce a human antibody or antigen-binding fragment thereof. In one case, the mouse is used to produce an antigen-binding protein selected from an antibody, a multispecific antibody (eg, a bispecific antibody), a scFv, a bis-scFV, a diabody, a triabody, a tetrabody, one V-NAR, one VHH, one VL, one F (ab), one F (ab) 2, one DVD (i.e. dual variable domain antigen binding protein), one SVD (i.e. binding protein a single variable domain antigen) or a bispecific T lymphocyte coupler (BiTE).

Also described is the use of the mouse as described herein for the production of a drug (eg, an antigen-binding protein), or for the manufacture of a sequence encoding a variable sequence of a drug (eg , an antigen-binding protein), is provided for the treatment of a human disease or disorder.

Any of the embodiments and aspects described herein can be used together with each other, unless otherwise indicated or evident from the context. Other embodiments will be apparent to those skilled in the art from a review of the following description.

Brief description of the figures

FIG. 1A shows a general illustration, not to scale, for the direct genomic replacement of approximately three megabases (Mb) of the mouse immunoglobulin heavy chain variable gene locus (closed symbols) with approximately one megabase (Mb) of the variable gene locus. immunoglobulin heavy chain protein (open symbols).

FIG. 1B shows a general illustration, not to scale, for the direct genomic replacement of approximately three megabases (Mb) of the mouse immunoglobulin k light chain variable gene locus (closed symbols) with approximately 0.5 megabases (Mb) of the first, or proximal, of two nearly identical repeats of the human immunoglobulin k light chain variable gene locus (open symbols).

FIG. 2A shows a detailed illustration, not to scale, for three initial stages (AC) for direct genomic replacement of the mouse immunoglobulin heavy chain variable gene locus resulting in deletion of all Vh, Dh and Jh gene segments. mouse and replacement with three human Vh, all human Dh and Jh gene segments. A targeting vector for the first insertion of human immunoglobulin heavy chain gene segments (3hVH BACvec) is shown with a 67 kb 5 'mouse homology arm, a selection cassette (open rectangle), a specific recombination site of the site (open triangle), a 145 kb human genomic fragment and an 8 kb 3 'mouse homology arm. Human immunoglobulin gene segments (open symbols) and mouse (closed symbols), additional selection cassettes (open rectangles) and site-specific recombination sites (open triangles) inserted from subsequent targeting vectors are shown.

FIG. 2B shows a detailed illustration, not to scale, for six additional steps (DI) for direct genomic replacement of the mouse immunoglobulin heavy chain variable gene locus resulting in insertion of 77 additional human Vh gene segments and deletion of the final selection cassette. A targeting vector is shown for insertion of additional human Vh gene segments (18hVH BACvec) for initial insertion of human heavy chain gene segments (3hVH-CRE hybrid allele) with a 20 kb 5 'mouse homology arm, a selection cassette (open rectangle), a 196 kb human genomic fragment and a 62 kb human homology arm that overlaps the 5 'end of the initial insertion of the human heavy chain gene segments shown with a site-specific recombination site (open triangle) located 5 'from human gene segments. Gene segments of human immunoglobulins (open symbols) and mouse (closed symbols) and additional selection cassettes (open rectangles) inserted by subsequent targeting vectors are shown.

FIG. 2C shows a detailed illustration, not to scale, for three initial stages (AC) for direct genomic replacement of the mouse immunoglobulin k light chain variable gene locus resulting in deletion of all Vk and Jk gene segments mouse (IgK-CRE Hybrid Allele). Selection cassettes (open rectangles) and site-specific recombination sites (open triangles) inserted from targeting vectors are shown.

FIG. 2D shows a detailed illustration, not to scale, for 5 additional steps (DH) for direct genomic replacement of the mouse immunoglobulin k light chain variable gene locus resulting in the insertion of all Vk and Jk gene segments in the proximal repeat and the deletion of the final selection cassette (Hybrid Allele 40hVKdHyg). Gene segments of human immunoglobulins (open symbols) and mouse (closed symbols) and additional selection cassettes (open rectangles) inserted by subsequent targeting vectors are shown.

FIG. 3A shows a general illustration of the locations of the quantitative PCR primer / probe sets (qPCR) for ES cell scanning for insertion of human heavy chain gene sequences and loss of mouse heavy chain gene sequences. . The scanning strategy in ES cells and mice for the first insertion of human heavy chain genes is shown with qPCR primer / probe sets for the deleted region (C and D "loss" probes), the inserted region (G probes and H of "hIgH") and flanking regions ("retention" probes A, B, B, E, and F) on an unmodified mouse chromosome (above) and a correctly targeted chromosome (below).

FIG. 3B shows a representative estimate of the probe copy number observed in precursor and modified ES cells for the first insertion of human immunoglobulin heavy chain gene segments. The number of probe copies observed for probes A to F was calculated as 2 / 2AACt. AACt is calculated as avg [ACt (sample) - medACt (control)] where ACt is the difference in Ct between the test and reference probes (between 4 and 6 reference probes according to the test). The term medACt (control) is the median ACt of multiple (> 60) non-targeted DNA samples from precursor ES cells. Each modified ES cell clone was assayed in six-fold. To calculate the copy number of the IgH G and H probes in the precursor ES cells, these probes were assumed to have a copy number of 1 in the modified ES cells and a Ct (cycle threshold) was used. in English, cycle treshold) maximum of 35 although no amplification was observed.

FIG. 3C shows a representative calculation of copy numbers for four mice of each genotype that were calculated similarly using only D and H probes. Wild-type mice: TS mice; Heterozygous mice for the first insertion of human immunoglobulin gene segments: HET mice; Mice homozygous for the first insertion of human immunoglobulin gene segments: Homo mice.

FIG. 4A shows an illustration of the three steps used for the construction of 3hVH BACvec by bacterial homologous recombination (BHR). Gene segments of human immunoglobulins (open symbols) and mouse (closed symbols), selection cassettes (open rectangles) and site-specific recombination sites (open triangles) inserted from targeting vectors are shown.

FIG. 4B shows pulsed field gel electrophoresis (PFGE) of three BAC clones (B1, B2 and B3) after Notl digestion. Markers M1, M2, and M3 are low-range, mid-range, and lambda ladder PFG markers, respectively (New England BioLabs, Ipswich, MA).

FIG. 5A shows a schematic illustration, not to scale, of sequential modifications of the mouse immunoglobulin heavy chain locus with increasing amounts of human immunoglobulin heavy chain gene segments. Homozygous mice were made from each of the three different stages of heavy chain humanization. The open symbols reflect the human sequence; the closed symbols reflect the mouse sequence.

FIG. 5B shows a schematic illustration, not to scale, of sequential modifications of the mouse immunoglobulin k light chain locus with increasing amounts of human immunoglobulin k light chain gene segments. Homozygous mice were made from each of the three different stages of humanization of the k light chain. The open symbols reflect the human sequence; the closed symbols reflect the mouse sequence.

FIG.6 shows FACS plot charts of B lymphocyte populations in humanized wild-type mice and VELOCIMMUNE®. Spleen cells (upper row, third row from upper and lower row) or inguinal lymph node (second row from top) of wild type (ts) or VELOCIMMUNE® 1 (V1), VELOCIMMUNE® were stained 2 (V2) or VELOCIm Mu NE® 3 (V3) were stained to determine B lymphocytes expressing surface IgM (top row and second row from top), surface immunoglobulin containing light chains ko A (third row from top) or surface IgM of specific haplotypes (bottom row) and populations separated by FACS.

FIG. 7A shows representative heavy chain CDR3 sequences of VELOCIMMUNE® antibodies randomly selected around the V ^h -D ^h -J ^h (CDR3) junction, demonstrating diversity of binding and nucleotide additions. Heavy chain CDR3 sequences are grouped according to the use of the D ^h gene segment, the germline of which is provided on each group in bold. The V ^h gene segments for each heavy chain CDR3 sequence are observed in parentheses at the 5 'end of each sequence (eg 3-72 is human V ^h 3-72). The J ^h gene segments for each heavy chain CDR3 are observed in parentheses at the 3 'end of each sequence (eg, 3 is human J ^h 3). The SEQ ID NOs for each sequence shown are as follows, from top to bottom: SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39. FIG. 7B shows representative light chain CDR3 sequences of randomly selected VELOCIMMUNE® antibodies around the Vk-Jk (CDR3) junction, demonstrating binding diversity and nucleotide additions. The Vk gene segments for each light chain CDR3 sequence are indicated in parentheses at the 5 'end of each sequence (eg 1-6 is human Vk1-6). The Jk gene segments for each light chain CDR3 are observed in parentheses at the 3 'end of each sequence (eg, 1 is human Jk1). The SEQ ID NOs for each sequence shown are as follows, from top to bottom: SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58.

FIG. 8 shows the somatic hypermutation frequencies of heavy and light chains of scored VELOCIMMUNE® antibodies (after alignment with paired germline sequences) as a percentage of sequences changed at each nucleotide position (NT; left column) or amino acid ( AA; right column) between sets of 38 (non-immunized IgM), 28 (non-immunized IgG), 32 (non-immunized IgG IgK), 36 (immunized IgG) or 36 (IgG immunized IgK) sequences. Shaded bars indicate the location of the CDRs.

FIG. 9A shows serum immunoglobulin levels for IgM and IgG isotypes in wild type mice (open bars) or VELOCIMMUNE® (closed bars).

FIG. 9B shows serum immunoglobulin levels for the IgA isotype in mice of the wild type (open bars) or VELOCIMMUNE® (closed bars).

FIG. 9C shows serum immunoglobulin levels for the IgE isotype in mice of the wild type (open bars) or VELOCIMMUNE® (closed bars).

FIG. 10A shows antigen-specific IgG titers against serum interleukin-6 receptor seven VELOCIMMUNE® (VI) mice and five wild-type (TS) mice after two (bleed 1) or three (bleed 2) rounds of immunization with Interleukin-6 receptor ectodomain.

FIG. 10B shows anti-interleukin-6 receptor specific IgG isotype titers from seven VELOCIMMUNE® (VI) mice and five wild-type (TS) mice.

FIG. 11A shows the affinity distribution of anti-interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® mice.

FIG. 11B shows antigen-specific blocking of anti-interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® (VI) and wild-type (TS) mice.

FIG. 12 shows a schematic illustration, not to scale, of mouse ADAM6a and ADAM6b genes at the mouse immunoglobulin heavy chain locus. A targeting vector (Target Vector mADAM6) used for insertion of mouse ADAM6a and ADAM6b into a humanized endogenous heavy chain locus is shown with a selection cassette (HYG: hygromycin) flanked by site-specific recombination sites (Frt ) which include engineered modified restriction sites at the 5 'and 3' ends.

FIG. 13 shows a schematic illustration, not to scale, of a human ADAM6 pseudogene (hADAM6 ^) located between the variable genetic segments of human heavy chain 1-2 (V ^h 1-2) and 6-1 (V ^h 6-1). . A targeting vector for bacterial homologous recombination (Target Vector hADAM6 ^) is shown to suppress a human ADAM6 pseudogene and insert unique restriction sites into a human heavy chain locus with a site-flanked (NEO: neomycin) cassette. site-specific recombination (loxP) sites including engineering modified restriction sites at the 5 'and 3' ends. An illustration, not to scale, of the resulting targeted humanized heavy chain locus containing a genomic fragment encoding the mouse ADAM6a and ADAM6b genes is shown, including a selection cassette flanked by site-specific recombination sites.

FIG. 14A shows FACS contour plots of selected lymphocytes in singlets for surface expression of IgM and B220 in the bone marrow for mice homozygous for human k and light heavy chain variable genes (H / k) loci and homozygous mice for light chain Heavy human k and human variable gene locus having an inserted mouse genomic fragment comprising mouse ADAM6 (H / k-A6) genes. The percentage of immature (B220intIgM +) and mature (B220highIgM +) B lymphocytes is observed in each contour graph.

FIG. 14B shows the total number of immature (B220intIgM +) and mature (B220highIgM +) B lymphocytes in the bone marrow isolated from femurs of mice homozygous for human heavy gene and light chain k (H / k) genes and mice homozygous for locus of human heavy and human k light chain variable genes having a mouse ectopic genomic fragment encoding mouse ADAM6 (H / k-A6) genes.

FIG. 15A shows FACS contour plots of selected CD19 + B lymphocytes for surface expression of c-kit and CD43 in the bone marrow for mice homozygous for human and human heavy light chain k (H / k) variable gene loci and mice homozygous for human k light and human heavy chain variable gene locus having a mouse ectopic genomic fragment encoding mouse ADAM6 (H / K-A6) genes. The percentage of pro-B (CD19 + CD43 + ckit +) and pre-B (CD19 + cD43'ck¡t) lymphocytes is observed in the upper right and lower left quadrants, respectively, of each contour graph.

FIG. 15B shows the total number of pro-B lymphocytes (CD19 + CD43 + ckit +) and pre-B lymphocytes (CD19 + CD43'ckit) in the bone marrow isolated from femurs of mice homozygous for human and light heavy chain variable gene loci human k (H / k) and mice homozygous for the human k light and human heavy chain variable gene locus having a mouse ectopic genomic fragment comprising mouse ADAM6 (H / k-A6) genes.

FIG. 16A shows FACS contour charts of selected lymphocytes in singlets for surface expression of CD19 and CD43 in the bone marrow for mice homozygous for human k light chain and H (k) variable gene loci and homozygous mice for k light chain. Heavy human and human variable gene locus having an ectopic mouse genomic fragment encoding mouse ADAM6 (H / k-A6) genes. The percentage of immature B lymphocytes (CD19 + c D43 ‘), pre-B (cDl9 + CD43int) and pro-B (CD19 + CD43 +) is observed in each contour graph.

FIG. 16B shows histograms of immature B lymphocytes (CD19 + CD43 ') and pre-B (CD19 + CD43int) in the bone marrow of mice homozygous for human k and light heavy chain variable gene loci (H / k) and homozygous mice. for human heavy and human k light chain variable gene loci having a mouse ectopic genomic fragment encoding mouse ADAM6 (h / k-A6) genes.

FIG. 17A shows FACS contour plots of selected lymphocytes in singlets for CD19 and CD3 surface expression in splenocytes for mice homozygous for human heavy chain and human k light chain (H / k) genes and homozygous mice for heavy human k light chain. and human variable gene locus having an ectopic mouse genomic fragment encoding mouse ADAM6 (H / k-A6) genes. The percentage of B (CD19 + CD3 ') and T (CD19'CD3 +) lymphocytes is observed in each contour graph.

FIG. 17B shows FAC contour plots for selected CD19 + B cells for surface expression of IgA and IgK light chain in the spleen of mice homozygous for human K light chain (H / k) variable genes and homozygous mice for human K light chain and human heavy chain variable gene loci having an ectopic mouse genomic fragment comprising mouse ADAM6 (H / K-A6) genes. The percentage of B lymphocytes lgA + (upper left quadrant) and lgK ° (lower right quadrant) is observed in each contour graph.

FIG. 17C shows the total number of CD19 + B lymphocytes in the spleen of mice homozygous for the human K light and human heavy chain variable gene locus (H / k) and mice homozygous for the human K light and human heavy chain variable gene loci that they have an ectopic mouse genomic fragment comprising mouse ADAM6 (H / K-A6) genes.

FIG. 18A shows FACS contour plots of selected CD19 + B lymphocytes for surface IgM expression in the spleen of mice homozygous for human K and light heavy chain variable genes (H / k) loci and mice homozygous for variable gene loci of human heavy and light chain human K having an ectopic mouse genomic fragment comprising mouse ADAM6 (H / k-A6) genes. The percentage of B lymphocytes (CD19 + IgDhighIgMint) is observed in each contour graph. The arrow in the right contour graph illustrates the maturation process of B lymphocytes in relation to IgM and IgD surface expression.

FIG. 18B shows the total number of B lymphocytes in the spleen of mice homozygous for human k light and human heavy chain variable gene locus (H / k) and mice homozygous for human k light and human heavy chain variable gene loci having an ectopic mouse genomic fragment encoding mouse ADAM6 (H / k-A6) genes during maturation from CD19 + IgMhighIgDint to CD19 + IgMintIgDhigh.

FIG. 19 illustrates a targeting strategy to replace endogenous mouse immunoglobulin light chain variable region gene segments with a human Vk1-39Jk5 gene region.

FIG. 20 illustrates a targeting strategy to replace endogenous mouse immunoglobulin light chain variable region gene segments with a human Vk3-20Jk1 gene region.

FIG. 21 illustrates a targeting strategy to replace endogenous mouse immunoglobulin light chain variable region gene segments with a human VpreB / JA5 gene region.

FIG. 22 shows the percentage of peripheral blood CD19 + B lymphocytes (y-axis) from wild-type (TS) mice, mice homozygous for an engineered human rearranged Vk1-39Jk5 light chain region (Vk1-39Jk5 HO), and homozygous mice for an engineered human rearranged Vk3-20Jk1 light chain region (Vk3-20Jk1 HO).

FIG. 23A shows the relative expression of mRNA (y-axis) of a light chain derived from Vk1-39 in a quantitative PCR assay using probes specific for binding of an engineered human rearranged Vk1-39Jk5 light chain region (Binding Probe Vk1-39Jk5) and the human Vk1-39 gene segment (probe Vk1-39) in a homozygous mouse for a replacement of the endogenous Vk and Jk gene segments with human Vk and Jk (Hk) gene segments, a wild type mouse ( TS) and a heterozygous mouse for an engineered human rearranged Vk1-39Jk5 light chain region (Vk1-39Jk5 HET). Signals are normalized to mouse Ck expression. N.D .: not detected.

FIG. 23B shows the relative expression of mRNA (y axis) of a light chain derived from Vk1-39 in a quantitative PCR assay using specific probes for binding of an engineered human rearranged Vk1-39Jk5 light chain region (Binding Probe Vk1-39Jk5) and the human Vk1-39 gene segment (probe Vk1-39) in a homozygous mouse for a replacement of the endogenous Vk and Jk gene segments with human Vk and Jk (Hk) gene segments, a wild type mouse ( TS) and a mouse homozygous for an engineered human rearranged Vk1-39Jk5 light chain region (Vk1-39Jk5 HO). Signals are normalized to mouse Ck expression.

FIG. 23A shows the relative expression of mRNA (y-axis) of a light chain derived from Vk3-20 in a quantitative PCR assay using probes specific for binding of an engineered human rearranged Vk3-20Jk1 light chain region (Binding Probe Vk3-20Jk1) and the human Vk3-20 gene segment (probe Vk3-20) in a homozygous mouse for a replacement of the endogenous Vk and Jk gene segments with human Vk and Jk (Hk) gene segments, a wild type mouse ( TS) and a heterozygous (HET) and homozygous (HO) mouse for an engineered rearranged human Vk3-20Jk1 light chain region. Signals are normalized to mouse Ck expression.

FIG.24A shows the IgM (left) and IgG (right) titer in wild type (TS; N = 2) and mice homozygous for an engineered human rearranged Vk1-39Jk5 light chain region (Vk1-39Jk5 HO; N = 2) immunized with p-galactosidase.

FIG. 24B shows the titer of total immunoglobulins (IgM, IgG, IgA) in wild type (TS; N = 5) and mice homozygous for an engineered human rearranged Vk3-20Jk1 light chain region (Vk3-20Jk1 HO; N = 5 ) immunized with p-galactosidase.

Detailed description

The term "antibody", as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V ^h ) and a heavy chain constant region (C ^h ). The heavy chain constant region comprises three domains, C ^h 1, C ^h 2, and C ^h 3. Each light chain comprises a light chain variable region (V ^l ) and a light chain constant region (C ^l ). Regions V ^H and V ^L can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions are conserved (FR). Each V ^h and V ^l comprise three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1 , HCDR2 and HCDR3, light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3 The term "high affinity" refers to an antibody that has a K ^d with respect to its target epitope of approximately 1 ° 9M or less (eg, about 1 x 1 ° '9 M, 1 x 1 °' 1 ° M, 1 x 10'11 M, or about 1 x 1 ° '12 M.) In one embodiment, K ^d is measured by resonance surface plasmon, eg BIACORE ™, in another embodiment, K ^d is measured by ELISA.

The phrase "bispecific antibody" includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two non-identical heavy chains, and each heavy chain specifically binds to a different epitope on two different molecules (eg, different epitopes on two different immunogens) or on the same molecule (for example, different epitopes on the same immunogen). If a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four or more orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. Epitopes specifically bound by the bispecific antibody may be on the same target or on a different target (eg, on the same protein or a different protein). Bispecific antibodies can be prepared, for example, by combining heavy chains that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same immunogen can be fused with nucleic acid sequences encoding the same or different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a C ^h 1 domain (N-terminal to C-terminal), a hinge, a C ^h 2 domain, and a C ^h 3 domain, and an immunoglobulin light chain that does not confer epitope binding specificity but that can be associated with each heavy chain, or that can be associated with each heavy chain and that can bind one or more of the epitopes linked by the epitope binding regions. of the heavy chain, or that can associate with each heavy chain and allow the binding of one or both heavy chains to one or both epitopes.

The term "cell" includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single-cell or multi-cell), bacterial cells (eg, E. coli strains , Bacillus spp., Streptomyces spp., Etc.), mycobacterial cells, fungal cells, yeast cells (eg S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (eg SF-9, SF-21, insect cells infected by baculovirus, Trichoplusia ni, etc.), cells from non-human animals, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (eg, CHO K1, DXB-11 CHO, Veggie-CHO), COS (eg, COS-7), retinal cell, Vero , CV1, kidney (eg HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (eg BHK21), Jurkat , Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell and a cell line derived from a previously mentioned cell. In some embodiments, the cell comprises one or more viral genes, eg, a retinal cell that expresses a viral gene (eg, a PER.C6 ™ cell).

The phrase "complementarity determining region", or the term "CDR", includes an amino acid sequence encoded by a nucleic acid sequence of immunoglobulin genes from an organism that normally (ie, in a wild-type animal) appears between two framework regions in a variable region of a light or heavy chain of an immunoglobulin molecule (eg, an antibody or a T-cell receptor). A CDR may be encoded by, for example, a germline sequence or a rearranged or unordered sequence, and, for example, by an untreated or mature B lymphocyte or by a T lymphocyte. A CDR may be somatically mutated for example, varying from a sequence encoded in the germline of an animal), humanized and / or modified with amino acid substitutions, additions or deletions. In some circumstances (eg, for a CDR3), CDRs may be encoded by two or more sequences (eg, germline sequences) that are not contiguous (eg, in an unordered nucleic acid sequence) but they are contiguous in a B lymphocyte nucleic acid sequence, eg, as a result of cutting and splicing or connecting the sequences (eg, VDJ recombination to form a heavy chain CDR3).

The term "conservative", when used to describe a conservative amino acid substitution, includes the substitution of one amino acid residue for another amino acid residue having a side chain R group with similar chemical properties (eg, charge or hydrophobicity ). In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, eg, the ability of a variable region to specifically bind to a target epitope with a desired affinity. Examples of amino acid groups having side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / arginine, lysine / arginine, alanine / valine, glutamate / aspartate, and asparagine / glutamine. In some embodiments, a conservative amino acid substitution can be a substitution of any natural residue in a protein with alanine, as used in, for example, alanine sweep mutagenesis. In some embodiments, a conservative substitution is made that has a positive value on the PAM250 logarithmic probability matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45. In some embodiments, the substitution is a moderately conservative substitution where the substitution has a non-negative value in the PAM250 logarithmic probability matrix.

In some embodiments, the positions of the residues on an immunoglobulin light or heavy chain differ by one or more conservative amino acid substitutions. In some embodiments, the positions of residues on an immunoglobulin light chain or a functional fragment thereof (eg, a fragment that allows expression and secretion of, eg, eg, a B lymphocyte) are not identical. to a light chain whose amino acid sequence is listed herein, but differs in one or more conservative amino acid substitutions.

The phrase "epitope binding protein" includes a protein that has at least one CDR and that is capable of selectively recognizing an epitope, eg, is capable of binding an epitope with a K ^d that is approximately one micromolar or less (for example, a K ^d that is about 1 x 10-6 M, 1 x 10-7 M, 1 x 10-9 M, 1 x 10-9 M, 1 x 10-10 M, 1 x 10-11 M , or approximately 1 x 10-12 M). Therapeutic epitope binding proteins (eg, therapeutic antibodies) often require a K ^d that is in the nanomolar or picomolar range.

The phrase "functional fragment" includes fragments of epitope-binding proteins that can express, secrete, and specifically bind to an epitope with a K ^d in the micromolar, nanomolar, or picomolar range. Specific recognition includes having a K ^d that is at least in the micromolar range, nanomolar range, or picomolar range.

The term "germline" includes reference to an immunoglobulin nucleic acid sequence in a somatically unmutated cell, eg, a B or pre-B lymphocyte or a hematopoietic cell not somatically mutated.

The phrase "heavy chain", or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain constant region sequence from any organism. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Heavy chain fragments include CDR, CDR, and FR and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a C ^h 1 domain, a hinge, a C ^h 2 domain and a C ^h 3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an epitope (eg, recognizing the epitope with a K ^d in the micromolar, nanomolar, or picomolar range), that is capable of being expressed and secreted from a cell, and that comprises at least one CDR.

The term "identity" when used in relation to sequence, includes identity determined by a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments described herein, identities are determined using a ClustalW v. Alignment. 1.83 (slow) using an open gap penalty of 10.0, gap extension penalty of 0.1 and using a Gonnet similarity matrix (MacVector ™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to the identity of the sequences will depend on the specific sequences, but in the case of a light chain constant domain, the length must contain a sequence of sufficient length to fold into a light chain constant domain. that is capable of associating with itself to form a canonical light chain constant domain, for example, capable of forming two beta sheets comprising beta strands and capable of interacting with at least one C ^h 1 domain of a human or mouse. In the case of the C ^h 1 domain, the length of the sequence must contain a sequence of sufficient length to fold into a C ^h 1 domain that is capable of forming two beta sheets comprising beta chains and that can interact with at least one domain light chain constant of a mouse or human.

The phrase "immunoglobulin molecule" includes two immunoglobulin heavy chains and two immunoglobulin light chains. Heavy chains can be identical or different, and light chains can be identical or different.

The phrase "light chain" includes an immunoglobulin light chain sequence from any organism and, unless otherwise specified, includes human k and A light chains and a VpreB, as well as surrogate light chains. Light chain variable domains (V ^l ) generally include three light chain CDRs and four conserved framework regions (FR, unless otherwise specified. Generally, a full length light chain includes, from the amino terminus to the terminus carboxyl, a V ^l including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a domain constant region light chain. light chains include those, for example, which can not bind selectively to a first or second epitope selectively bound by the epitope-binding protein they appear in. Light chains also include those that bind and recognize, or assist the heavy chain to bind and recognize, one or more epitopes selectively bound by the protein binding to epitope in which they appear.

Universal light chains, or common light chains, refers to light chains produced in mice as described herein, where mice are highly restricted in selecting available gene segments to produce a light chain variable domain. As a result, such mice produce a light chain derived from, in one embodiment, no more than one or two unreordered light chain V segments and no more than one or two unreordered light chain J segments (eg, a V and one J, two V and one J, one V and two J, two V and two J). In one embodiment, no more than one or two rearranged light chain V / J sequences, per Example, a rearranged human Vk1-39Jk5 sequence or a rearranged human V k3-20Jk1 sequence. In various embodiments, the universal light chains include somatically mutated versions (eg, affinity matured).

The phrase "somatically mutated" includes a reference to a nucleic acid sequence of a B lymphocyte that has undergone a class change, wherein the nucleic acid sequence of an immunoglobulin variable region (eg, a heavy chain variable domain or which includes a heavy chain CDR or FR sequence) in the B-lymphocyte with class change is not identical to the nucleic acid sequence in the B-lymphocyte before the class change, such as, for example, a difference in a CDR nucleic acid sequence or framework between a B lymphocyte that has not undergone a class change and a B lymphocyte that has undergone a class change. "Somatically mutated" includes a reference to affinity matured B lymphocyte nucleic acid sequences that are not identical to the corresponding immunoglobulin variable region sequences in B lymphocytes that are not affinity matured (that is, sequences in the genome of germline cells). The phrase "somatically mutated" also includes a reference to a nucleic acid sequence of the immunoglobulin variable region of a B lymphocyte after exposure of the B lymphocyte to an epitope of interest, where the nucleic acid sequence differs from the sequence of corresponding nucleic acid before exposure of the B lymphocyte to the epitope of interest. The phrase "somatically mutated" refers to antibody sequences that have been generated in an animal, eg, a mouse that has human immunoglobulin variable region nucleic acid sequences, in response to immunogen exposure, and that result from the selection processes inherently operational in such an animal.

The term "non-rearranged", with respect to nucleic acid sequences, includes nucleic acid sequences that exist in the germline of an animal cell.

The phrase "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) comprising the following amino acid regions, in N-terminal to C-terminal sequence (unless otherwise noted opposite): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Mice with Humanized Immunoglobulin Locus

The mouse as a genetic model has been greatly enhanced by transgenic and gene suppression technologies, which have allowed the study of the effects of targeted overexpression or deletion of specific genes. Despite all of its advantages, the mouse still has genetic hurdles that make it an imperfect model for human disease and an imperfect platform for testing or making human therapeutic agents. First, although approximately 99% of human genes have a mouse homolog (Waterston, RH, et. Al. (2002). Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520-562.), Frequently, potential therapeutic agents either fail to cross-react or achieve inadequate cross-reaction, with mouse orthologs of predicted human targets. To avoid this problem, selected target genes can be "humanized," that is, the mouse gene can be deleted and replaced by the corresponding human orthologous gene sequence (eg, US 6,586,251, US 6,596. .541 and US document 7,105,348). Initially, efforts to humanize mouse genes using a "more transgenic gene deletion humanization" strategy involved crossing a mouse bearing a deletion (ie nulligenic) of the endogenous gene with a mouse carrying a randomly integrated human transgene ( see, for example, Bril, WS, et al. (2006). Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg (593) to Cys substitution. Thromb Haemost 95, 341-347; Homanics, GE , et al. (2006). Production and characterization of murine models of classic and intermediate maple syrup urine disease. BMC Med Genet 7, 33; Jamsai, D., et al. (2006). A humanized BAC transgenic / knockout mouse model for HbE / beta-thalassemia. Genomics 88 (3): 309-15; Pan, Q., et al. (2006). Different role for mouse and human CD3delta / epsilon heterodimer in preT cell receptor (preTCR) function: human CD3delta / epsilon heterodimer restores the defective preTCR function in CD3 gamma- and CD3 gammade lta-deficient mice. Mol Immunol 43, 1741-1750). But those efforts were hampered by size limitations; Conventional gene suppression technologies were not sufficient to directly replace genes from large mice with their large human genomic counterparts. A simple direct homologous replacement approach is rarely attempted, in which an endogenous mouse gene is directly replaced by the human homologous gene at the same precise genetic location of the mouse gene (i.e., at the endogenous mouse locus), because to technical difficulties. Until now, direct replacement efforts have included elaborate and complicated procedures, limiting the length of genetic material that can be handled and the precision with which it can be manipulated.

Exogenously introduced human immunoglobulin transgenes are rearranged on precursor B lymphocytes in mice (Alt, FW, Blackwell, TK, and Yancopoulos, GD (1985). Immunoglobulin genes in transgenic mice. Trends Genet 1, 231-236). This finding was exploited by engineered mice using the more transgenic gene deletion approach to express human antibodies (Green, LL et al. (1994). Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 7, 13-21; Lonberg, N. (2005). Human antibodies from transgenic animals. Nat Biotechnol 23, 1117-1125; Lonberg, N., et al. (1994). Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368, 856 859; Jakobovits, A., et al. (2007). From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice. Nat Biotechnol 25, 1134-1143). The heavy chain and light chain k loci of Endogenous mouse immunoglobulin were inactivated in these mice by targeted deletion of small but critical portions of each endogenous locus, followed by introduction of human immunoglobulin gene loci as large randomly integrated transgenes, as described above or as minichromosomes (Tomizuka, K., et al. (2000) Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci USA 97, 722-727). Such mice represented an important advance in genetic engineering; fully human monoclonal antibodies isolated from them produced a promising therapeutic agent to treat a variety of human diseases (Gibson, TB, et al. (2006). Randomized phase III trial results of panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, in metastatic colorectal cancer. Clin Colorectal Cancer 6, 29-31; Jakobovits et al., 2007; Kim, YH, et al. (2007). Clinical efficacy of zanolimumab (HuMax-CD4): two Phase II studies in refractory cutaneous T-cell lymphoma. Blood 109 (11): 4655-62; Lonberg, 2005; Maker, AV, et al. (2005). Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase I / II study. Ann Surg Oncol 12, 1005-1016; McClung, MR, et al. (2006). Denosumab in postmenopausal women with low bone mineral density. N Engl J Med 354, 821-831). But, as discussed above, these mice show compromised development of B lymphocytes and immune deficiencies compared to wild-type mice. Such problems potentially limit the ability of mice to withstand an intense humoral response and, consequently, to generate fully human antibodies against some antigens. Deficiencies may be due to: (1) ineffective functionality due to random introduction of human immunoglobulin transgenes and resulting incorrect expression due to lack of upstream and downstream control elements (Garrett, FE, et al. (2005 Chromatin architecture near a potential 3 'end of the high locus involves modular regulation of histone modifications during B-Cell development and in vivo occupancy at CTCF sites. Mol Cell Biol 25, 1511-1525; Manis, JP, et al. ( Elucidation of a downstream boundary of the 3 'IgH regulatory region. Mol Immunol 39, 753-760; Pawlitzky, I., et al. (2006). Identification of a candidate regulatory element within the 5' flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A. J Immunol 176, 6839-6851); 2) inefficient species-to-species interactions between human constant domains and mouse components of the B-cell receptor signaling complex on the cell surface, which may impair the signaling processes necessary for normal B-cell maturation, proliferation, and survival (Hombach , J., et al. (1990) Molecular components of the B-cell antigen receptor complex of the IgM class. Nature 343, 760-762); and (3) ineffective inter-species interactions between soluble human immunoglobulins and mouse Fc receptors that could reduce affinity selection (Rao, SP, et al. (2002). Differential expression of the inhibitory IgG Fc receptor FcgammaRIIB on germinal center cells: implications for selection of high-affinity B cells. J Immunol 169, 1859 1868) and immunoglobulin serum concentrations (Brambell, FW, et al. (1964). A Theoretical Model of Gamma-Globulin Catabolism. Nature 203, 1352-1354; Junghans , RP, and Anderson, CL (1996) .The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor. Proc Natl Acad Sci USA 93, 5512-5516; Rao et al., 2002; Hjelm, F ., et al. (2006). Antibody-mediated regulation of the immune response. Scand J Immunol 64, 177-184; Nimmerjahn, F., and Ravetch, JV (2007). Fc- receptors as regulators of immunity. Adv Immunol 96, 179-204). These deficiencies can be corrected by humanizing in situ only the variable regions of the mouse immunoglobulin loci within their natural locations at the endogenous heavy and light chain loci. This would effectively result in mice producing reverse chimeric antibodies (i.e., human V: mouse C) that would be capable of normal interactions and selection with the mouse environment based on preservation of mouse constant regions. Additionally, such reverse chimeric antibodies are readily redrawn into fully human antibodies for therapeutic purposes.

A method is described for a large in situ genetic replacement of mouse germline immunoglobulin variable genes with human germline immunoglobulin variable genes while maintaining the ability of mice to generate offspring. Specifically, the precise replacement of six megabases of mouse immunoglobulin heavy chain and light chain k variable gene loci with their human counterparts is described while leaving the mouse constant regions intact. As a result, mice have been created that have an accurate replacement of their entire repertoire of germline immunoglobulins with equivalent variable sequences of human germline immunoglobulins, while maintaining constant mouse regions. Human variable regions are linked to mouse constant regions to form human-mouse chimeric immunoglobulin loci that are rearranged and expressed at physiologically appropriate levels. The expressed antibodies are "reverse chimeras", that is, they comprise human variable region sequences and mouse constant region sequences. These mice that have humanized immunoglobulin variable regions that express antibodies that have human variable regions and mouse constant regions are called humanized VELCOIMMUNE® mice.

Humanized VELOCIMMUNE® mice display a fully functional humoral immune system that is essentially indistinguishable from that of wild-type mice. They show normal cell populations at all stages of B lymphocyte development. They show normal morphology of the lymphoid organs. The antibody sequences from humanized VELOCIMMUNE® mice show normal rearrangement of variable segments and normal somatic hypermutation. Antibody populations in these mice reflect isotype distributions resulting from the normal class change (eg, cis change from normal isotype). Immunization of humanized VELOCIMMUNE® mice results in potent humoral responses that generate a wide variety of antibodies that have suitable human immunoglobulin variable domains as candidates therapeutic This platform provides an abundant source of affinity matured human immunoglobulin variable region sequences to produce antibodies and other pharmaceutically acceptable antigen binding proteins.

It is the precise replacement of the variable sequences of mouse immunoglobulins with variable sequences of human immunoglobulins that allows the production of humanized VELOCIMMUNE® mice. However, even precise replacement of the endogenous mouse immunoglobulin sequences at heavy and light chain loci with equivalent human immunoglobulin sequences, by sequential recombination of very large stretches of human immunoglobulin sequences, can present certain challenges due to evolution divergence of the immunoglobulin loci between mouse and man. For example, the intergenic sequences interspersed within the immunoglobulin loci are not identical between mice and humans and, in some circumstances, may not be functionally equivalent. Differences between mice and humans at their immunoglobulin loci can still result in abnormalities in humanized mice, particularly when certain portions of endogenous mouse immunoglobulin heavy chain loci are humanized or manipulated. Some modifications at the mouse immunoglobulin heavy chain loci are deleterious. Harmful modifications may include, for example, loss of the ability of the modified mice to mate and produce offspring.

A large-scale, in situ replacement of six megabases of the variable regions of the mouse immunoglobulin heavy and light chain loci (V ^h -D ^h -J ^h and Vk-Jk) was performed with human genomic sequences. corresponding 1.4 megabase, while leaving the flanking mouse sequences intact and functional within the hybrid loci, including all mouse constant chain genes and transcriptional control locus regions (Figure 1). Specifically, the human V ^h , D ^h , J ^h , V k, and Jk gene sequences were introduced by the gradual insertion of 13 chimeric BAC targeting vectors carrying overlapping fragments of the human germline variable loci into ES cells. of mouse using VELOCIGENE® genetic engineering technology (see, eg, US Patent No. 6,586,251 and Valenzuela, DM, et al. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21.652-659).

The humanization of mouse immunoglobulin genes represents the largest genetic modification of the mouse genome to date. While previous efforts with randomly integrated human immunoglobulin transgenes have had some success (discussed above), direct replacement of mouse immunoglobulin genes with their human counterparts dramatically increases the efficiency with which fully human antibodies can be generated effective in normal mice. Additionally, such mice display a dramatically increased diversity of fully human antibodies that can be obtained after immunization with virtually any antigen, compared to mice bearing disabled endogenous loci and fully human antibody transgenes. Multiple versions of replaced humanized loci show completely normal levels of mature and immature B lymphocytes, compared to mice with randomly integrated human transgenes, which show significantly reduced B lymphocyte populations at various stages of differentiation. While efforts to increase the number of human gene segments in human transgenic mice have reduced such defects, expanded immunoglobulin repertoires have not fully corrected for reductions in B lymphocyte populations compared to wild-type mice.

Despite the almost wild-type humoral immune function observed in mice with replaced immunoglobulin loci, there are other challenges encountered in employing a direct immunoglobulin replacement that is not found in some approaches employing randomly integrated transgenes. Differences in the genetic makeup of the immunoglobulin loci between mice and humans has led to the discovery of beneficial sequences for the reproduction of mice with replaced immunoglobulin gene segments. Specifically, mouse ADAM genes located within the endogenous immunoglobulin locus are optimally present in mice with replaced immunoglobulin loci, due to their role in fertility.

Genomic Location and Function of Mouse ADAM6

Male mice lacking the ability to express any functional ADAM6 protein show a serious defect in the mice's ability to mate and generate offspring. Mice lack the ability to express a functional ADAM6 protein by replacing all or substantially all mouse immunoglobulin variable region gene segments with human variable region gene segments. The loss of ADAM6 function is due to the ADAM6 locus being located within a region of the endogenous mouse immunoglobulin heavy chain variable region gene locus, close to the 3 'end of the V ^h gene segment locus that is upstream of gene segments D ^h . In order to breed mice that are homozygous for a replacement of all or substantially all endogenous mouse heavy chain variable gene segments with human heavy chain variable gene segments, it is generally a cumbersome approach to configure males and females to be homozygous for replacement and awaiting productive mating. Successful litters are relatively rare, and the average litter size is very low. Instead, heterozygous males have been used for replacement to mate with homozygous females for replacement to generate progeny that are heterozygous for replacement, and then a homozygous mouse is reared from it. The present inventors have determined that the probable cause of loss of fertility in male mice is the absence in homozygous male mice of a functional ADAM6 protein.

The ADAM6 protein is a member of the ADAM family of proteins, where ADAM is an acronym for A Disintegrin And Metalloprotease (A Desintegrin and Metalloprotease). The ADAM family of proteins is large and diverse, with diverse functions. Some members of the ADAM family are involved in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of the protein fertiline, which is involved in sperm-egg interactions. ADAM3, or ciritestina, seems necessary for the union of the spermatozoa to the zona pellucida. The absence of ADAM2 or ADAM3 results in infertility. ADAM2, ADAM3 and ADAM6 have been postulated to form a complex on the surface of mouse sperm.

The human ADAM6 gene, normally located between the V ^h 1-2 and V ^h 6-1 segments of the human V ^h gene, appears to be a pseudogene (Figure 12). In mice, there are two ADAM6 genes, ADAM6a and ADAM6b that are located in an intergenic region between the mouse V ^h and D ^h gene segments, and in the mouse, genes a and b are oriented in a transcriptional orientation opposite to the transcriptional orientation. of the surrounding immunoglobulin gene segments (Figure 11). In mice, a functional ADAM6 locus appears to be needed for normal fertilization. A functional ADAM6 locus or sequence, then, refers to an ADAM6 locus or sequence that can complement, or rescue, the drastically reduced fertilization shown in male mice with missing or damaged endogenous ADAM6 loci.

The position of the intergenic sequence in mice encoding ADAM6a and ADAM6b renders the intergenic sequence susceptible to modification by modifying an endogenous mouse heavy chain. When the V ^h gene segments are deleted or replaced, or when the D ^h gene segments are deleted or replaced, there is a high probability that a resulting mouse exhibits a severe fertility deficit. To compensate for the deficit, the mouse is modified to include a nucleotide sequence that encodes a protein that will complement the loss in ADAM6 activity due to a modification of the endogenous mouse ADAM6 locus. In various embodiments, the complementary nucleotide sequence is one that encodes a mouse ADAM6a, a mouse ADAM6b, or a homolog or an ortholog or a functional fragment thereof that rescues fertility deficit.

The nucleotide sequence that salvages fertility can be placed in any suitable ectopic position. It can be placed in any suitable ectopic position in the genome. In one embodiment, the nucleotide sequence can be introduced into a transgene that is randomly integrated into the mouse genome. In one embodiment, the sequence can be maintained episomically, that is, on a separate nucleic acid rather than on a mouse chromosome. Suitable positions include positions that are transcriptionally permissive or active, eg, a ROSA26 locus.

The term "ectopic" is intended to include a shift, or a location at a position that is not normally found in nature (for example, the location of a nucleic acid sequence at a position that is not the same position that it is found at nucleic acid sequence in a wild type mouse). The term in various embodiments is used in the sense that its target is outside its normal or appropriate position. For example, the phrase "an ectopic nucleotide sequence that encodes ..." refers to a nucleotide sequence that appears at a position where it is not normally found in the mouse. For example, in the case of an ectopic nucleotide sequence encoding a mouse ADAM6 protein (or an ortholog or a homolog or a fragment thereof that provides the same or similar fertility benefit in male mice), the sequence may be placed at a different position in the mouse genome than is normally found in a wild type mouse. A mouse ADAM6 functional homolog or ortholog is a sequence that confers a salvage of loss of fertility (eg, loss of the ability of a male mouse to generate offspring by mating) that is observed in an ADAM6 '/ mouse _. Functional homologs or orthologs include proteins that have at least about 89% or more identity, for example, up to 99% identity, with the amino acid sequence of ADAM6a and / or with the amino acid sequence of ADAM6b, and that can complement or rescue the ability to successfully mate, from a mouse that has a genotype that includes a gene deletion or deletion of ADAM6a and / or ADAM6b.

The ectopic position can be anywhere (for example, as with the random insertion of a transgene containing a mouse ADAM6 sequence), or it can be, for example, in an approaching position (but is not exactly the same) to its location in a wild-type mouse (eg, at a modified endogenous mouse immunoglobulin locus, but upstream or downstream of its natural position, eg, within a modified immunoglobulin locus but between different gene segments, or at a different position in a mouse RV intergenic sequence). An example of an ectopic localization is localization within a humanized immunoglobulin heavy chain locus. For example, a mouse comprising a replacement of one or more endogenous V ^h gene segments with human V ^h gene segments, where the replacement removes an endogenous ADAM6 sequence, may be engineered to have a mouse ADAM6 sequence located within the sequence containing the human V ^h gene segments. The resulting modification would generate a mouse ADAM6 (ectopic) sequence within a human gene sequence, and the (ectopic) location of the sequence Mouse ADAM6 within the human gene sequence may approximate the position of the human ADAM6 pseudogene (ie, between two V segments) or may approximate the position of the mouse ADAM6 sequence (ie, within the VD intergenic region) .

In various aspects, mice can be produced comprising deletions or replacements of the endogenous heavy chain variable region locus or portions thereof that contain an ectopic nucleotide sequence encoding a protein that confers fertility benefits similar to mouse ADAM6 (by example, an ortholog or homolog or a fragment thereof that is functional in a male mouse). The ectopic nucleotide sequence may include a nucleotide sequence encoding a protein that is an ADAM6 homolog or ortholog (or fragment thereof) of a different mouse strain or a different species, eg, a different rodent species , and that confers a fertility benefit, for example, greater number of litters during a specific period of time, and / or greater number of offspring per litter, and / or the ability of a sperm of a male mouse to cross an oviduct mouse to fertilize a mouse egg.

In one embodiment, ADAM6 is a homolog or ortholog that is at least 89% to 99% identical to a mouse ADAM6 protein (eg, at least 89% to 99% identical to mouse ADAM6a or mouse ADAM6b ). In one embodiment, the ectopic nucleotide sequence encodes one or more independently selected proteins of a protein at least 89% identical to mouse ADAM6a, a protein at least 89% identical to mouse ADAM6b, and a combination thereof. In one embodiment, the homolog or ortholog is a guinea pig rat, hamster, mouse or pig protein that is or is modified to be approximately 89% or more identical to mouse ADAM6a and / or mouse ADAM6b. In one embodiment, the homolog or ortholog is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to mouse ADAM6a and / or ADAM6b of mouse.

ADAM6 Ectopic in Mice with Humanized Heavy Chain

Mice that produce human antibodies have been available for some time. Although they represent an important advance in the development of human therapeutic antibodies, these mice show a series of significant abnormalities that limit their usefulness. For example, they show compromised development of B lymphocytes. Compromised development may be due to a variety of differences between transgenic mice and wild-type mice.

Human antibodies may not optimally interact with mouse pre-B or lymphocyte receptors on the surface of mouse cells that indicate maturation, proliferation, or survival during clonal selection. Fully human antibodies may not optimally interact with a mouse Fc receptor system; mice express Fc receptors that do not show a one-to-one correspondence with human Fc receptors. Finally, several mice that produce fully human antibodies do not include all of the genuine mouse sequences, eg, enhancer elements and other downstream locus control elements, that may be necessary for the development of wild-type B lymphocytes.

Mice that produce fully human antibodies generally comprise endogenous immunoglobulin loci that are somewhat inactivated, and human transgenes that comprise variable and constant immunoglobulin gene segments are introduced at a random location in the mouse genome. As long as the endogenous locus is inactivated enough not to rearrange the gene segments to form a functional immunoglobulin gene, the goal of producing fully human antibodies in said mouse can be achieved, although with the compromised development of B lymphocytes.

Although it is obliged to produce fully human antibodies from the human transgenic locus, the generation of human antibodies in a mouse appears to be an unfavorable process. In some mice, the process is so unfavorable that it results in the formation of chimeric heavy chains, human variables / mouse constants (but not light chains) through the trans shift mechanism. By this mechanism, transcripts encoding fully human antibodies undergo the isotype change in trans from the human isotype to a mouse isotype. The process is in trans, because the fully human transgene is located apart from the endogenous locus that conserves an undamaged copy of a mouse heavy chain constant region gene. Although the trans change is easily evident in such mice, the phenomenon is still insufficient to rescue the development of B lymphocytes, which remains frankly affected. In any case, the antibodies with trans change produced in said mice conserve completely human light chains, since the phenomenon of trans change does not appear to occur with respect to light chains; the change in trans is likely based on endogenous locus change sequences used (albeit differently) on the normal cis- changed isotype . Therefore, even when mice engineered to produce fully human antibodies select a trans-switching mechanism to produce antibodies with mouse constant regions, the strategy remains insufficient to rescue normal development of B lymphocytes.

A primary concern in the production of antibody-based human therapeutic agents is to make a sufficiently large diversity of human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize and bind to specific epitopes with an affinity. desirable, generally, but not always, with high affinity. Before the development of the humanized VELOCIMMUNE® mice, there was no indication that mice expressing human variable regions with mouse constant regions showed significant differences from mice that produced human antibodies from a transgene. That assumption, however, was incorrect.

The humanized VELOCIMMUNE® mice, which contain a precise replacement of mouse immunoglobulin variable regions with human immunoglobulin variable regions at endogenous mouse loci, exhibit remarkable similarity to wild-type mice in the development of B lymphocytes. surprising and astonishing development, humanized VELOCIMMUNE® mice showed an essentially normal wild-type response to immunization that differed only in a significant way from wild-type mice, the variable regions generated in response to immunization are fully human.

Humanized VELOCIMMUNE® mice contain a large-scale, precise replacement of the immunoglobulin heavy chain (IgH) germline and mouse immunoglobulin light chain (eg, k, IgK) light chain variable regions with the variable regions of corresponding human immunoglobulin, at the endogenous loci. In total, approximately six megabases of mouse loci are replaced with approximately 1.4 megabases of human genomic sequence. This precise replacement results in a mouse with hybrid immunoglobulin loci producing heavy and light chains that have human variable regions and a mouse constant region. Precise replacement of the mouse V ^h -D ^h -J ^h and Vk-Jk segments leaves flanking mouse sequences intact and functional at the hybrid immunoglobulin loci. The mouse's humoral immune system works like that of a wild-type mouse. The development of B lymphocytes is not impeded in any significant respect and a rich diversity of human variable regions is generated in the mouse upon exposure to the antigen.

Humanized VELOCIMMUNE® mice are possible because the immunoglobulin gene segments for heavy and light chains k are rearranged similarly in humans and mice, which is not to say that their loci are the same or even clearly not are. However, the loci are similar enough that humanization of the heavy chain variable gene locus can be achieved by replacing approximately 3 million base pairs of contiguous mouse sequence containing all V ^h , D ^h gene segments, and J ^h with approximately 1 million bases of contiguous human genomic sequence basically covering the equivalent sequence of a human immunoglobulin locus.

In some embodiments, the additional replacement of certain sequences of genes of the constant region of mouse with human gene sequences (for example, replacing the sequence C ^h 1 mouse sequence C ^h 1 human and replacement of the sequence C ^l mouse with human ^l C sequence) gives as result mice locus hybrid immunoglobulins produce antibodies having human variable regions and human constant regions partially suitable for, for example, produce fragments of fully human antibodies, for example, Fab completely human. Mice with hybrid immunoglobulin loci show normal variable gene segment rearrangement, normal somatic hypermutation, and normal class change. These mice display a humoral immune system that is indistinguishable from that of wild-type mice and exhibit normal cell populations at all stages of development of B lymphocytes and normal lymphoid organ structures, even in cases where mice lack a complete repertoire. of gene segments of human variable region. Immunization of these mice results in strong humoral responses that exhibit a wide diversity of use of variable gene segments.

Precise replacement of the mouse germline variable region gene segments enables mice to be produced that have partially human immunoglobulin loci. Because partially human immunoglobulin loci are rearranged, hypermutated, and class changed in a normal manner, partially human immunoglobulin loci generate antibodies in a mouse comprising human variable regions. Nucleotide sequences encoding variable regions can be identified and cloned, then fused (eg, in an in vitro system ) with any sequence of choice, eg, any immunoglobulin isotype suitable for a particular use, resulting in an antibody. or an antigen-binding protein derived entirely from human sequences.

Large-scale humanization by recombinant engineering methods was used to modify mouse embryonic stem cells (ES) to precisely replace up to 3 megabases of the mouse immunoglobulin heavy chain locus that included essentially all V ^h , D gene segments. mouse ^h and J ^h with equivalent human gene segments with a human genomic sequence of up to 1 megabase containing some or essentially all of the human V ^h , D ^h and J ^h gene segments. Up to a 0.5 megabase segment of the human genome comprising one of the two repeats encoding essentially all human Vk and Jk gene segments was used to replace a 3 megabase segment of the mouse immunoglobulin k light chain locus containing essentially all mouse Vk and Jk gene segments.

Mice with such replaced immunoglobulin loci may comprise a disruption or deletion of the endogenous mouse ADAM6 locus, which typically lies between the V ^h plus 3 'gene segment and the D ^h plus 5' gene segment at the chain locus. mouse immunoglobulin weighing. Disruption in this region can lead to the reduction or elimination of functionality of the endogenous mouse ADAM6 locus. If the segments Vh genes plus 3 'from the human heavy chain repertoire are used in a replacement, an intergenic region containing a pseudogene that appears to be a human ADAM6 pseudogene is present between these Vh gene segments, i.e. between Vh1-2 and Vh1-6 humans. However, male mice comprising this human intergenic sequence show little or no fertility.

Mice are described which comprise the replaced loci as described above, and which also comprise an ectopic nucleic acid sequence encoding a mouse ADAM6, where the mice show essentially normal fertility. In one embodiment, the ectopic nucleic acid sequence is SEQ ID NO: 3, located between human Vh1-2 and Vh1-6 at the modified endogenous mouse heavy chain locus. The transcription direction of the ADAM6 genes of SEQ ID NO: 3 is opposite to the transcription direction of the surrounding human Vh gene segments. Although the examples herein show fertility rescue by placing the ectopic sequence between the indicated human Vh gene segments, experts will recognize that the location of the ectopic sequence is expected at any suitable transcriptionally permissive locus in the mouse genome ( or even extrachromosomally) similarly rescue fertility in a male mouse.

The phenomenon of complementing a mouse lacking a functional ADAM6 locus with an ectopic sequence comprising a mouse ADAM6 gene or an ortholog or a homolog or a functional fragment thereof is a general method that is applicable to the rescue of any mouse with locus ADAM6 endogenous non-functional or minimally functional. Therefore, a large number of mice comprising a disruptive modification of ADAM6 from the immunoglobulin heavy chain locus can be rescued with the compositions and methods of the invention. Accordingly, the invention encompasses mice with a wide variety of immunoglobulin heavy chain locus modifications that compromise the endogenous function of ADAM6 present ectopically. Some (non-limiting) examples are provided in the present description. In addition to the described VELOCIMMUNE® humanized mice, the compositions and methods related to ADAM6 can be used in many applications, for example, when modifying a heavy chain locus in a wide variety of ways.

In one aspect, there is provided a mouse comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or a homolog or a functional fragment thereof), a replacement for all or substantially all mouse Vh gene segments with one or more human Vh gene segments, a replacement of all or substantially all mouse Dh gene segments and mouse Jh gene segments with human Dh and human Jh gene segments; where the mouse lacks a Ch1 and / or hinge region. In one embodiment, the mouse produces a unique variable domain binding protein that is a dimer of immunoglobulin chains selected from: (a) human Vh-mouse Ch1-mouse Ch2-mouse Ch3; (b) human Vh-mouse hinge-mouse Ch2-mouse Ch3; and, (c) human Vh-mouse Ch2-mouse Ch3.

In one aspect, the fertility rescue nucleotide sequence is placed within a human immunoglobulin heavy chain variable region sequence (eg, between human Vh1-2 and Vh1-6 gene segments) in a mouse having a replacement of all or substantially all mouse immunoglobulin heavy chain variable gene segments (mVH, mDH and itiJh) with one or more human immunoglobulin heavy chain variable gene segments (hVH, hDH, and hJH) and the mouse further comprises a replacement of all or substantially all mouse immunoglobulin k light chain variable gene segments (mVK, mJK) with one or more human immunoglobulin k light chain variable gene segments (hVK and hJK). In one embodiment, the nucleotide sequence is placed between a human Vh1-2 gene segment and a human Vh1-6 gene segment in a humanized VELOCIMMUNE® mouse (US 6,596,541 and US 7,105,348). In one embodiment, the VELOCIMMUNE® humanized mouse thus modified comprises a replacement with all or substantially all of the human immunoglobulin heavy chain variable gene segments (all hVH, hDH and hJH) and all or substantially all of the gene segments k light chain variables of human immunoglobulins (hVk and hJk).

In one aspect, a functional mouse ADAM6 locus (or an ortholog or a homolog or a functional fragment thereof) can be placed in the middle of human Vh gene segments that replace Vh gene segments from endogenous mice. In one embodiment, all or substantially all of the mouse Vh gene segments are removed and replaced with one or more human Vh gene segments, and the mouse ADAM6 locus is positioned immediately adjacent to the 3 'end of the human Vh gene segments, or between two human Vh gene segments. In a specific embodiment, the mouse ADAM6 locus is placed between two Vh gene segments near the 3 'terminus of the inserted human Vh gene segments. In a specific embodiment, the replacement includes human Vh1-2 and Vh6-1 gene segments, and the mouse ADAM6 locus is positioned downstream of the Vh1-2 gene segment and upstream of the Vh6-1 gene segment. In a specific embodiment, the arrangement of the human Vh gene segments is then as follows (upstream to downstream with respect to the direction of transcription of the human Vh gene segments): human Vh1-2 - mouse ADAM6 locus - Vh6 -1 human. In a specific embodiment, the ADAM6 pseudogene between human Vh1-2 and human Vh6-1 is replaced with the mouse ADAM6 locus. In one embodiment, the orientation of one or more mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite with respect to the direction of transcription compared to the orientation of the human Vh gene segments. Alternatively, the mouse ADAM6 locus can be placed in the intergenic region between the human Vh plus 3 'gene segment and the Dh plus 5' gene segment. This may be the case, if the segment Dh plus 5 'is mouse or human.

Similarly, a mouse modified with one or more human Vl gene segments (eg, Vk or VA segments) that replaces all or substantially all endogenous mouse Vh gene segments can be modified to maintain the mouse ADAM6 locus. endogenously, as described above, for example, employing a targeting vector having a downstream homology arm that includes a mouse ADAM6 locus or a functional fragment thereof, or to replace a damaged mouse ADAM6 locus with an ectopic sequence positioned between two human Vl gene segments or between human Vl gene segments and a Dh gene segment (either human or mouse, eg VA m / hDH), or a J gene segment (either human or mouse, for example, Vk Jh). In one embodiment, the replacement includes two or more human Vl gene segments, and the mouse ADAM6 locus or a functional fragment thereof is placed between the two plus 3 'Vl gene segments. In a specific embodiment, the arrangement of the human Vl gene segments is then as follows (upstream to downstream with respect to the direction of transcription of the human gene segments): human Vl3'-1-mouse ADAM6 locus- Vl3 ' human. In one embodiment, the orientation of one or more mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite with respect to the direction of transcription compared to the orientation of the human Vl gene segments. Alternatively, the mouse ADAM6 locus can be positioned in the intergenic region between the human Vl plus 3 'gene segment and the Dh plus 5' gene segment. This may be the case if the Dh plus 5 'segment is mouse or human.

In one aspect, a mouse is described with a replacement of one or more endogenous mouse Vh gene segments, and comprising at least one endogenous mouse Dh gene segment. In said a mouse, modification of the endogenous mouse Vh gene segments may comprise a modification of one or more of the Vh plus 3 'gene segments, but not of the Dh plus 5' gene segment, where care is taken so that the modification of the one or more Vh plus 3 'gene segments do not interrupt or render the endogenous mouse ADAM6 locus non-functional. For example, in one case, the mouse comprises a replacement of all or substantially all endogenous mouse Vh gene segments with one or more human Vh gene segments, and the mouse comprises one or more endogenous Dh gene segments and an ADAM6 locus of functional endogenous mouse.

[Removed]

The use of mice containing an ectopic sequence encoding a mouse ADAM6 protein or an ortholog or a functional homolog or homolog thereof is useful when the modifications interrupt the function of endogenous mouse ADAM6. The probability of disrupting the function of endogenous mouse ADAM6 is high when modifications are made at the mouse immunoglobulin loci, particularly when modifying the mouse immunoglobulin heavy chain variable regions and surrounding sequences. Therefore, such mice provide a particular benefit when mice are produced with immunoglobulin heavy chain loci that are fully or partially deleted, fully or partially humanized, or replaced (eg, with Vk sequences). or VA) in whole or in part. Methods of producing the described genetic modifications for the mice described below are known to those skilled in the art.

Mice containing an ectopic sequence that encodes a mouse ADAM6 protein, or a substantially identical or similar protein that confers the fertility benefits of a mouse ADAM6 protein, are particularly useful in conjunction with modifications of a locus of the variable region gene of mouse immunoglobulin heavy chain that disrupts or deletes the endogenous mouse ADAM6 sequence. Although primarily described in relation to mice expressing antibodies with human variable regions and mouse constant regions, such mice are useful in connection with any genetic modification that disrupts the endogenous mouse ADAM6 gene. It will be recognized by experts that this encompasses a wide variety of genetically engineered mice that contain modifications of the mouse immunoglobulin heavy chain variable region gene locus. These include, for example, mice with a deletion or replacement of all or a part of the mouse immunoglobulin heavy chain gene segments, regardless of other modifications.

Genetically modified mice comprising a mouse, rodent or other ectopic ADAM6 gene (or an ortholog or homolog or fragment) functional in a mouse, and one or more immunoglobulin variable and / or constant region gene segments are disclosed. human.

A mouse is described comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein, a replacement of all or substantially all of the mouse Vh gene segments with one or more human Vh gene segments; a replacement of all or substantially all of the mouse Dh gene segments with one or more human Dh gene segments; and a replacement of all or substantially all of the mouse Jh gene segments with one or more human JH gene segments.

In one case, the mouse further comprises replacing a mouse Ch1 nucleotide sequence with a human Ch1 nucleotide sequence. In one embodiment, the mouse further comprises replacing a mouse hinge nucleotide sequence with a human hinge nucleotide sequence. In one case, the mouse further comprises replacing an immunoglobulin light chain variable locus (Vl and Jl) with a variable light chain locus of human immunoglobulin. In one case, the mouse further comprises replacing a mouse immunoglobulin light chain constant region nucleotide sequence with a human immunoglobulin light chain constant region nucleotide sequence. In a specific case, Vl, Jl and Cl are immunoglobulin k light chain sequences. In one specific case, the mouse comprises a mouse Ch2 and mouse Ch3 immunoglobulin constant region sequence fused with a human hinge and a human Ch1 sequence, so that the mouse immunoglobulin loci are rearranged to form a gene encoding a binding protein comprising (a) a heavy chain having a human variable region, a human Ch1 region, a human hinge region and a mouse Ch2 region and a mouse Ch3 region; and (b) a gene encoding an immunoglobulin light chain comprising a human variable domain and a human constant region.

A mouse is described comprising an ectopic ADAM6 sequence encoding a functional ADAM6 protein, a replacement of all or substantially all mouse Vh gene segments with one or more human Vl gene segments, and optionally a replacement of all or substantially all the Dh gene segments and / or Jh gene segments with one or more human Dh gene segments and / or human Jh gene segments, or optionally a replacement of all or substantially all Dh gene segments and JH gene segments with one or more gene segments JL human.

A mouse is described comprising a replacement of all or substantially all mouse Vh, Dh and Jh gene segments with one or more Vl gene segments, one or more Dh and one or more J (eg Jk or JA), wherein the gene segments are operably linked to an endogenous mouse hinge region, wherein the mouse forms a rearranged immunoglobulin chain gene containing, from 5 'to 3' in the direction of transcription, human Vl-human Dh or mouse -J human or mouse - mouse hinge -Ch2 mouse - Ch3 mouse. In one case, region J is a human Jk region. In one case, region J is a human JH region. In one case, region J is a human JA region. In one case, the human VL region is selected from a human VA region and a human Vk region.

In specific cases, the mouse expresses a single variable domain antibody having a mouse or human constant region and a variable region derived from a human Vk, a human Dh, and a human Jk; a human Vk, a human Dh and a human Jh; a human VA, a human Dh and a human JA; a human VA, a human Dh and a human Jh; a human Vk, a human Dh and a human JA; a human VA, a human Dh and a human Jk. In a specific case, the recombination recognition sequences are modified to allow productive rearrangements to occur between enumerated V, D and J gene segments or between enumerated V and J gene segments.

In one aspect, the mouse provided comprises an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or a homolog or a functional fragment thereof), a replacement of all or substantially all mouse Vh gene segments with one or more human Vl gene segments, a replacement of all or substantially all Dh gene segments and mouse Jh gene segments with human Jl gene segments; where the mouse lacks a Ch1 and / or hinge region.

In one embodiment, the mouse lacks a sequence encoding a Ch1 domain. In one embodiment, the mouse lacks a sequence encoding a hinge region. In one embodiment, the mouse lacks a sequence encoding a Ch1 domain and a hinge region.

In a specific embodiment, the mouse expresses a binding protein comprising a human immunoglobulin light chain variable domain (A or k) fused to a mouse Ch2 domain that is bound to a mouse Ch3 domain.

In one aspect, the mouse provided comprises an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or a homolog or a functional fragment thereof), a replacement of all or substantially all mouse Vh gene segments with one or more human Vl gene segments, a replacement of all or substantially all mouse Dh and Jh gene segments with human Jl gene segments.

In one embodiment, the mouse comprises a deletion of an immunoglobulin heavy chain constant region gene sequence encoding a Ch1 region, a hinge region, a Ch1 region and a hinge, or a Ch1 region and a hinge region and a Ch2 region. .

In one embodiment, the mouse produces a single variable domain binding protein comprising a homodimer selected from the following: (a) human Vl-mouse Ch1-mouse Ch2-mouse Ch3; (b) human Vl-mouse hinge-mouse Ch2-mouse Ch3; (c) Human Vl- Mouse Ch2- Mouse Ch3.

In one aspect, there is provided a mouse with an inactivated endogenous immunoglobulin heavy chain locus, comprising an inactivated or deleted endogenous mouse ADAM6 locus, wherein the mouse comprises a nucleic acid sequence expressing a human or mouse antibody or human / mouse or other chimeric antibody. In one embodiment, the nucleic acid sequence is present in an integrated transgene that integrates randomly in the mouse genome. In one embodiment, the nucleic acid sequence is in an episome (eg, a chromosome) that is not found in a wild type mouse.

Common or Universal Light Chain

Previous efforts to produce useful multispecific epitope binding proteins, for example, bispecific antibodies, have been hampered by a variety of problems that frequently share a common paradigm: in vitro selection or manipulation of sequences to be rationally engineered, or to engineer by trial and error, a suitable format to match a heterodimeric bispecific human immunoglobulin. Unfortunately, most, if not all, in vitro engineering approaches provide largely ad hoc solutions that are suitable, if at all, for individual molecules. On the other hand, no in vivo methods have been performed to employ complex organisms to select appropriate pairings that are capable of leading to human therapeutic agents.

Generally, natural mouse sequences are often not a good source of human therapeutic agent sequences. For at least that reason, the generation of mouse immunoglobulin heavy chain variable regions that pair with a common human light chain is of limited practical utility. More in vitro engineering efforts would be made in a trial and error process to try to humanize the variable sequences of the mouse heavy chain while hoping to preserve the specificity and affinity of the epitope while maintaining the ability to couple with the common human light chain, with uncertain results. At the end of such a process, the final product may retain some of the specificity and affinity, and be associated with the common light chain, but ultimately, immunogenicity in a human would likely remain a deep risk.

Therefore, a mouse suitable for making human therapeutic agents would include a suitably large repertoire of human heavy chain variable region gene segments instead of endogenous mouse heavy chain variable region gene segments. Gene segments of the human heavy chain variable region should be able to be rearranged and recombined with an endogenous mouse heavy chain constant domain to form a reverse chimeric heavy chain (i.e., a heavy chain comprising a human variable domain and constant region mouse). The heavy chain must be capable of class change and somatic hypermutation so that a large repertoire of suitable heavy chain variable domains is available for the mouse to select one that can be associated with the limited repertoire of human light chain variable regions.

A mouse that selects a common light chain for a plurality of heavy chains has practical utility. In various embodiments, antibodies expressing in a mouse that can only express one common light chain will have heavy chains that can associate and be expressed with an identical or substantially identical light chain. This is particularly useful in the production of bispecific antibodies. For example, such a mouse can be immunized with a first immunogen to generate a B lymphocyte that expresses an antibody that specifically binds to a first epitope. The mouse (or genetically the same mouse) can be immunized with a second immunogen to generate a B lymphocyte that expresses an antibody that specifically binds to the second epitope. Heavy chain variable regions can be cloned from B lymphocytes and expressed with the same heavy chain constant region and the same light chain and expressed in a cell to produce a bispecific antibody, where the light chain component of the bispecific antibody has selected by mouse to associate and express with the same light chain component.

The present inventors have engineered a mouse to generate immunoglobulin light chains that will adequately pair with a fairly diverse family of heavy chains, including heavy chains whose variable regions separate from the germline sequences, eg, the regions variables affinity matured or somatically mutated. In various embodiments, the mouse is designed to pair human light chain variable domains with human heavy chain variable domains that comprise somatic mutations, thereby allowing a route to high affinity binding proteins suitable for use as human therapeutic agents.

The genetically engineered mouse, through the long and complex process of antibody selection within an organism, makes biologically appropriate decisions by pairing a diverse collection of human heavy chain variable domains with a limited number of human light chain options. In order to accomplish this, the mouse is engineered to present a limited number of human light chain variable domain options along with a wide variety of human heavy chain variable domain options. Upon exposure with an antigen, the mouse maximizes the number of solutions in its repertoire to develop an antibody to the antigen, largely or solely limited by the number or light chain options in its repertoire. In various embodiments, this includes allowing the mouse to achieve adequate and compatible somatic mutations of the light chain variable domain, which will, however, be compatible with a relatively large variety of human heavy chain variable domains, including in particular heavy chain variable domains somatically mutated human.

To achieve a limited repertoire of light chain options, the mouse is engineered to do no functionally or substantially non-functional its ability to produce or rearrange, a natural mouse light chain variable domain. This can be accomplished, for example, by deleting gene segments from the mouse light chain variable region. The endogenous mouse locus can then be modified by suitable exogenous human light chain variable region gene segments, operably linked to the endogenous mouse light chain constant domain, such that exogenous human variable region gene segments can be combined with the exogenous mouse light chain constant region gene and form a rearranged reverse chimeric light chain gene (human variable, mouse constant). In various embodiments, the variable region of the light chain is capable of being somatically mutated. In various embodiments, to maximize the ability of the light chain variable region to acquire somatic mutations, appropriate enhancers are preserved in the mouse. For example, by modifying a mouse k light chain locus to replace endogenous mouse k light chain gene segments with human k light chain gene segments, the mouse intron k enhancer and the mouse 3'k enhancer are maintained, or are not functionally interrupted.

The genetically engineered mouse can express a limited repertoire of reverse chimeric light chains (human variable, mouse constant) associated with a variety of reverse chimeric heavy chains (human variable, mouse constant). In various embodiments, the endogenous mouse k light chain gene segments are deleted and replaced with a single (or two) rearranged human light chain region (s), operably linked to the endogenous mouse C ^k gene. In embodiments to maximize somatic hypermutation of the rearranged human light chain region, the mouse intronic k enhancer and the mouse 3'k enhancer are maintained. In various embodiments, the mouse also comprises a non-functional light chain A locus, or a deletion thereof or a deletion that renders the locus incapable of producing a light chain A.

The modified mouse engineered provided may comprise, in various embodiments, a locus variable light chain region lacking gene segments V ^L and J ^L light chain endogenous mouse and comprising a variable region of light chain human reformatted in one embodiment, a rearranged human V ^l / J ^l sequence, operably linked to a mouse constant region, where the locus is capable of undergoing somatic hypermutation, and where the locus expresses a light chain comprising the V ^l / sequence J ^l human bound to a constant region of mouse. Therefore, in various embodiments, the locus comprises a mouse 3'k enhancer, which correlates with a normal or wild-type level of somatic hypermutation.

The genetically engineered mouse in various embodiments when immunized with an antigen of interest generates B lymphocytes showing a variety of rearrangements of human immunoglobulin heavy chain variable regions that are expressed and function with one or two rearranged light chains, including those Embodiments where the one or both light chains comprise human light chain variable regions comprising, for example, 1 to 5 somatic mutations. In various embodiments, the human light chains thus expressed are capable of associating and expressing themselves with any human immunoglobulin heavy chain variable region expressed in the mouse.

Epitope Binding Proteins that Bind More Than One Epitope

The compositions and methods described herein can be used to produce binding proteins that bind to more than one epitope with high affinity, eg, bispecific antibodies. Advantages of the invention include the ability to properly select for high binding heavy chain immunoglobulin chains (eg, affinity matured), each of which will associate with a single light chain.

The synthesis and expression of bispecific binding proteins has been problematic, in part because of problems associated with identifying a suitable light chain that can associate and express with two different heavy chains, and in part because of isolation problems. The methods and compositions described herein allow a genetically modified mouse to select, by natural processes, a suitable light chain that can associate and express itself with more than one heavy chain, including heavy chains that are somatically mutated (eg, matured by affinity). The sequences of V ^l and V ^h human B lymphocytes suitable immunized mice as described in present expressing matured antibodies affinity with chimeric heavy chain reverse (ie, human variable and constant mouse) can be identified and in-frame cloning into an expression vector with a suitable human constant region gene sequence (eg, human IgG1). Two such constructs can be prepared, where each construct encodes a human heavy chain variable domain that binds to a different epitope. One of the human V ^L (for example, human or Vk3-20Jk1 Vk1-39Jk5 human) in germline sequence or a B cell where sequence somatically mutated, can be fused in frame to a human constant region gene suitable (eg, a human k constant gene). These three fully human heavy and light constructs can be placed in a cell suitable for expression. The cell will express two main species: a homodimeric heavy chain with the identical light chain, and a heterodimeric heavy chain with the identical light chain. To allow for easy separation of these major species, one of the heavy chains is modified to omit a protein A binding determinant, resulting in a differential affinity for a homodimeric binding protein from a heterodimeric binding protein. . Compositions and methods that address this problem are described in USSN 12 / 832,838, filed on June 25, 2010, titled "Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format," published as US 2010 / 0331527A1.

An epitope binding protein is described, wherein the human Vl and Vh sequences are derived from mice described herein that have been immunized with an antigen comprising an epitope of interest.

An epitope binding protein is described comprising a first and a second polypeptide, the first polypeptide comprising, from the N-terminus to the C-terminus, a first epitope-binding region that selectively binds to a first epitope, followed by a region constant comprising a first Ch3 region of a human IgG selected from IgG1, IgG2, IgG4 and a combination thereof; and, a second polypeptide comprising, from the N-terminus to the C-terminus, a second epitope-binding region that selectively binds to a second epitope, followed by a constant region comprising a second Ch3 region of a human IgG selected from IgG1 , IgG2, IgG4 and a combination thereof, wherein the second Ch3 region comprises a modification that reduces or eliminates the binding of the second CH3 domain to protein A.

In one case, the second Ch3 region comprises an H95R modification (by IMGT exon numbering; H435R by EU numbering). In another embodiment, the second Ch3 region further comprises a Y96F modification (for IMGT; Y436F for EU).

In one case, the second Ch3 region is derived from a modified human IgG1 and further comprises a modification selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU).

In one case, the second Ch3 region is derived from a modified human IgG2 and further comprises a modification selected from the group consisting of N44S, K52N and V82I (IMGT; N384S, k 392N and V422I according to EU).

In one case, the second Ch3 region is derived from a modified human IgG4 and further comprises a modification selected from the group consisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT; Q355R, N384S, K392N, V397M, R409K , E419Q and V422I according to EU).

One method of producing an epitope-binding protein that binds more than one epitope is to immunize a first mouse according to the invention with an antigen comprising a first epitope of interest, wherein the mouse comprises a variable region locus. endogenous immunoglobulin light chain enzyme that does not contain an endogenous mouse Vl that is capable of rearranging and forming a light chain, wherein the locus of the endogenous mouse immunoglobulin light chain variable region is a single rearranged human Vl region operably linked to the gene from the endogenous mouse light chain constant region, and the rearranged human Vl region is selected from a human Vk1-39Jk5 and a human Vk3-20Jk1, and the endogenous mouse Vh gene segments have been totally or partially replaced with gene segments Human Vh, so that the immunoglobulin heavy chains produced by the mouse are uniquely or substantially heavy chains comprising human variable domains and d constant mouse omissions. When immunized, said mouse will produce a reverse chimeric antibody, which will comprise only one of the two human light chain variable domains (eg, one of human Vk1-39Jk5 or human Vk3-20Jk1). Once a B lymphocyte encoding a Vh is identified that binds to the epitope of interest, the nucleotide sequence of the Vh (and optionally the Vl) can be recovered (eg, by PCR) and cloned into a construct of in-frame expression with a suitable human immunoglobulin constant domain. This process can be repeated to identify a second Vh domain that binds to a second epitope, and a second Vh gene sequence can be recovered and cloned into an expression vector in-frame with a suitable second constant immunoglobulin domain. The first and second immunoglobulin constant domains can be the same or a different isotype, and one of the immunoglobulin constant domains (but not the other) can be modified as described herein or in US 2010 / 0331527A1, and the epitope binding protein can be expressed in a suitable cell and isolated based on its differential affinity for protein A compared to a homodimeric epitope binding protein, for example, as described in US 2010 / 0331527A1.

A method of producing a bispecific antigen-binding protein is described, which comprises identifying a first affinity matured human Vh (Vh1) nucleotide sequence (eg, comprising one or more somatic hypermutations) from a mouse as described in Herein, identify a second affinity matured human VH (VH2) nucleotide sequence (eg, comprising one or more somatic hypermutations) from a mouse as described herein, clone Vh1 in-frame with a strand human heavy lacking a determinative modification of protein A as described in US 2010 / 0331527A1 to form heavy chain 1 (HC1), clone Vh2 in-frame with a human heavy chain comprising a determinant of protein A as described in US 2010 / 0331527A1 to form heavy chain 2 (HC2), introduce an expression vector comprising HC1 and the same or different expression vector n comprising HC2 in a cell, wherein the cell also expresses a human immunoglobulin light chain comprising a human Vk1-39 / human JK5 or a human Vk3-20 / human JKl fused to a constant domain of the human light chain, allowing the cell to express a bispecific epitope binding protein comprising a Vh1-encoded Vh domain and a Vh domain encoded by Vh2, and isolating the bispecific epitope binding protein based on its differential ability to bind protein A compared to a monospecific homodimeric epitope binding protein. In a specific embodiment, HC1 is an IgG1, and HC2 is an IgG1 that comprises the H95R (IMGT; H435R for EU) modification and further comprises the Y96F (IMGT; Y436F for EU) modification. In one embodiment, the Vh1-encoded Vh domain, the Vh2-encoded Vh domain, or both, are somatically mutated.

Human Vh Genes Expressed with a Common Human Vl

A variety of human affinity mature antibody variable regions generated against four different antigens were expressed with their cognate light chain, or at least one of a human light chain selected from human Vk1-39Jk5, human Vk3-20Jk1, or human VpreBJA, 5 (see Example 10). For antibodies against each of the antigens, somatically mutated high affinity heavy chains from different gene families were successfully paired with rearranged human germline Vk1-39Jk5 and Vk3-20Jk1 regions and secreted from cells expressing the heavy and light chains. For Vk1-39Jk5 and Vk3-20Jk1, the Vh domains derived from the following human Vh gene families were favorably expressed: 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3- 11,3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31,4-39, 4-59, 5-51, and 6-1 . Therefore, a mouse engineered to express a limited repertoire of human Vl domains from one or both of Vk1-39Jk5 and Vk3-20Jk1 will generate a diverse population of somatically mutated human Vh domains from a modified Vh locus to replace segments. mouse VH gene sequences with human VH gene segments.

Mice engineered to express reverse chimeric immunoglobulin heavy chains (human variable, mouse constant) associated with a single rearranged light chain (eg, a Vk1-39 / J or a Vk3-20 / J), when they immunized with an antigen of interest, generated B lymphocytes comprising a variety of human Vh rearrangements, and expressed a variety of high-affinity antigen-specific antibodies with various properties with respect to their ability to block binding of the antigen to its ligand, and regarding its ability to bind to antigen variants (see Examples 14 to 15).

Therefore, the mice and methods described herein are useful for producing and selecting human immunoglobulin heavy chain variable domains, including somatically mutated human heavy chain variable domains, which are the result of a variety of rearrangements, which show a wide variety of affinities (including a Kd of about one nanomolar or less), a wide variety of specificities (including binding to different epitopes of the same antigen), and that are associated and expressed with the same or substantially the same human immunoglobulin light chain variable region.

A first mouse comprising a humanized heavy chain variable region locus can be paired with a second mouse comprising a nucleic acid sequence encoding a common or universal light chain locus as described herein. The first or second mouse may comprise an ectopic nucleic acid sequence encoding a mouse ADAM6 or an ortholog or a homolog or a functional fragment thereof. Progeny are bred to obtain mice homozygous for a humanized heavy chain locus, and homozygous for the universal light chain locus. The first mouse or the second mouse may comprise a modification of an endogenous mouse light chain locus to render the endogenous mouse light chain locus non-functional (eg, a deletion or gene deletion of, eg, a endogenous locus A and / ok). The first mouse may comprise a replacement of all or substantially all functional endogenous mouse V, D, and J gene segments with one or more unreordered human V, D, and J gene segments (eg, all or substantially all functional human V, D, and J gene segments); and the mouse comprises a replacement of all or substantially all functional light chain V and J gene segments with no more than one or no more than two rearranged light chain V / J sequences. The first mouse further comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 or an ortholog or a homolog or a functional fragment thereof. In one case, the ectopic nucleic acid sequence is at a humanized immunoglobulin heavy chain locus.

In one case, mice comprising the ectopic sequence and homozygous for the universal light chain locus and the humanized heavy chain locus are immunized with an antigen of interest to generate antibodies comprising a plurality of somatically mutated human variable domains that are associated and expressed with a universal light chain. In one case, the human heavy chain variable domain nucleic acid sequences identified in the mouse are employed in an expression system to produce a fully human antibody comprising the human heavy chain variable domain and a light chain comprising a mouse universal light chain sequence.

The following examples are provided to describe those skilled in the art how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the present inventors consider their invention. Attempts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperature, etc.) but some errors and experimental deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is indicated in degrees Celsius, and pressure is atmospheric or near atmospheric.

Examples

Example I

Humanization of Mouse Immunoglobulin Genes

Mouse and human bacterial artificial chromosomes (BACs) were used to engineer 13 different BAC targeting vectors (BACvec) for the humanization of mouse immunoglobulin heavy chain and light chain k loci. Tables 1 and 2 set forth detailed descriptions of the steps carried out for the construction of all the BACvecs used for the humanization of the mouse immunoglobulin heavy chain and light chain k chains, respectively.

Identification of human and mouse BAC.

Mouse BACs spanning the 5 'and 3' ends of the immunoglobulin k light chain and heavy chain loci were identified by hybridization of BAC library-stained filters or by PCR scanning DNA clusters of the library mouse BAC. Filters were hybridized under standard conditions using probes that corresponded to the regions of interest. Library groups were screened by PCR using unique primer pairs flanking the target region of interest. Additional PCR was performed using the same primers to deconvolve a given well and isolate the corresponding BAC of interest. Both BAC filters and library clusters were generated from 129 SvJ mouse ES cells (Incyte Genomics / lnvitrogen). Human BACs covering all immunoglobulin k light chain and heavy chain loci were identified by hybridization of filters stained with the BAC library (Caltech B, C or D libraries and RPCI-11 library, Research Genetics / lnvitrogen) a by scanning groups of the human BAC library (Caltech library, Invitrogen) using a PCR-based method or using a BAC final sequence database (Caltech D library, TIGR).

Construction of BACvec (Tables 1 and 2).

Bacterial homologous recombination (BHR) was performed as described (Valenzuela et al., 2003; Zhang, Y., et al. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20, 123-128). In most cases, linear fragments were generated by binding PCR-derived homology boxes to cloned cassettes, followed by gel isolation of binding products and electroporation in BHR-competent bacteria harboring the target BAC. After selection on the appropriate antibiotic Petri dishes, correctly recombined BACs were identified by PCR through both new junctions, followed by restriction analysis on pulsed field gels (Schwartz, DC, and Cantor, CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67-75) and spot verification by PCR using primers distributed across human sequences.

A 3hVH BACvec was constructed using three sequential BHR steps for the initial humanization step of the immunoglobulin heavy chain locus (FlG. 4A and Table 1). In the first step (Step 1), a cassette was introduced into a human precursor BAC upstream of the human V ^h 1-3 gene segment containing a region of homology to the mouse immunoglobulin heavy chain locus (HB1), a gene that confers resistance to kanamycin in bacteria and resistance to G418 in animal cells (kanR) and a site-specific recombination site (eg, loxP). In the second step (Step 2), a second cassette was introduced just downstream of the last segment J ^h containing a second region of homology to the mouse immunoglobulin heavy chain locus (HB2) and a gene that confers resistance to spectomycin in bacteria (specR). This second step included deletion of human immunoglobulin heavy chain locus sequences downstream of J ^h 6 and the chloramphenicol resistance gene of the BAC vector (cmR). In the third stage (Stage 3), the doubly modified human BAC (B1) was linearized using I-Ceul sites that had been added during the first two stages and integrated into a mouse BAC (B2) by BHR through the two regions of homology (HB1 and h B2). Drug selections for the first (cm / kan), second (spec / kan), and third (cm / kan) steps were designed to be specific to the desired products. Modified BAC clones were analyzed by pulsed gel electrophoresis (PFGE) after restriction enzyme digestion to determine the appropriate construction (FIG. 4B).

Similarly, an additional 12 BACvecs were engineered for the humanization of the heavy chain and light chain k loci. In some cases, binding of bAc instead of BHR was performed to co-link two large BACs by introducing rare restriction sites in both BHR precursor BACvec along with careful placement of selectable markers. This allowed for the survival of the desired binding product after selection with specific combinations of drug markers. Recombinant BACs obtained by binding after digestion with rare restriction enzymes were identified and explored similarly to those obtained by BHR (as described above).

Table 1

(continuation)

Table 2

Modification of embryonic stem cells (ES by their silgas in English) and generation of mice.

ES cell targeting (F1H4) was performed using the VELOCIGENE® genetic engineering method as described (Valenzuela et al., 2003). Obtaining mice from blastocyst modified ES cells (Valenzuela et al., 2003) or by injection of 8cell (Poueymirou et al., 2007) was as described. ES cells and targeted mice were confirmed by DNA scanning of ES cells or mice with unique sets of probes and primers in a PCR based assay (eg, FIG. 3A, 3B, and 3C). All studies in mice were supervised and approved by the Regeneron Institutional Animal Care and Use Committee (IACUC).

Karyotype Analysis and Fluorescent In Situ Hybridization (FISH).

Karyotype analysis was performed using Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). FISH was performed on targeted ES cells as described (Valenzuela et al., 2003). Probes corresponding to mouse BAC DNA or human BAC DNA were labeled by notch translation (Invitrogen) with the orange or green spectrum of fluorescently labeled dUTP nucleotides (Vysis).

Immunoglobulin Heavy Chain Variable Gene Locus.

Humanization of the variable region of the heavy chain locus was accomplished in nine sequential steps by direct replacement of approximately three million base pairs (Mb) of contiguous mouse genomic sequence containing all V ^h , D ^h, and gene segments. J ^h with approximately one Mb of contiguous human genomic sequence containing human equivalent gene segments (FIG. 1A and Table 1) using VELOCIGENE® genetic engineering technology (see, eg, US Patent 6,586,251 and Valenzuela et al., 2003).

The intron between J ^h gene segments and constant region genes (the JC intron) contains a transcriptional enhancer (Neuberger, MS (1983) Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. EMBO J 2, 1373-1378) followed of a region of simple repeats necessary for recombination during isotype change (Kataoka, T. et al. (1980) Rearrangement of immunoglobulin gamma 1-chain gene and mechanism for heavy-chain class switch. Proc Natl Acad Sci USA 77, 919-923). The junction between the human V ^h -D ^h -J ^h region and the mouse C ^h region (the proximal junction) was chosen to maintain the mouse heavy chain intronic enhancer and to change the domain to preserve both efficient expression and class change of the humanized heavy chain locus within the mouse. The exact nucleotide position of this and subsequent junctions in all replacements was possible using the VELOCIGENE® genetic engineering method ( supra), which employed bacterial homologous recombination activated by synthesized oligonucleotides. Therefore, the proximal junction was placed approximately 200 bp downstream of the last gene segment J ^h and the distal junction was placed several hundred upstream of the V ^h plus 5 'gene segment of the human locus and approximately 9 kb downstream of the mouse V ^h 1-86 gene segment, also known as J558.55. The mouse V ^h 1-86 (J558.55) gene segment is the most distal heavy chain variable gene segment, reported to be a pseudogene in C57BL / 6 mice, but potentially active, albeit with poor RSS sequence, at the directed 129 allele. Reportedly, the distal end of the mouse heavy chain locus may contain control elements that regulate locus expression and / or rearrangement (Pawlitzky et al., 2006).

A first insertion of the human immunoglobulin DNA sequence was achieved in the mouse using 144 kb of the proximal end of the human heavy chain locus containing 3 human V ^h gene segments, the 27 D ^h and 9 J ^h inserted at the proximal end from the mouse IgH locus, with a concomitant deletion of 16.6 kb of mouse genomic sequence, using approximately 75 kb of mouse homology arms (Step A, FIG. 2A; Tables 1 and 3, 3hVH). This large 144 kb insert and the accompanying 16.6 kb deletion was performed in a single step (Step A) that occurred at a frequency of 0.2% (Table 3). Correctly targeted ES cells were scored by a natural allele loss assay (LONA) (Valenzuela et al., 2003) using probes within and flanking the deleted mouse sequence and within the inserted human sequence, and the integrity of the human large insert using multiple probes spanning the entire insert (FIG. 3A, 3B and 3C). Because many rounds of sequential targeting of ES cells were anticipated, ES cell clones targeting this, and all subsequent steps, underwent karyotype analysis ( supra) and only those clones showing karyotypes were used for subsequent steps normal in at least 17 out of 20 extensions.

The targeted ES cells from Step A were re-targeted with a BACvec that produced a 19 kb deletion at the distal end of the heavy chain locus (Step B, FIG. 2A). The BACvec from Stage B contained a hygromycin resistance gene (hig) as opposed to the neomycin resistance gene (neo) contained in the BACvec from Stage A. The resistance genes of the two BACvecs were designed in such a way that , after successful redirection to the same chromosome, approximately three Mb of the mouse heavy chain variable gene locus containing all mouse V ^h gene segments other than V ^h 1-86 and all D ^h gene segments other than DQ52 , as well as the two resistance genes, were flanked by loxP sites; DQ52 and all segments of the mouse J ^h chain gene were deleted in Step A. Clones of ES cells doubly targeted on the same chromosome were identified by driving the proximal 3hVH cassette to homozygosity in high G418 (Mortensen, RM et al. al. (1992) Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol 12: 2391-2395) and following the fate of the distal hyg cassette. Mouse segments up to four Mb in size, which have been modified to be flanked by loxP sites, have been successfully suppressed in ES cells by transient expression of CRE recombinase with high efficiencies (up to "11%) even in the absence of drug selection (Zheng, B., et al. (2000). Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol Cell Biol 20: 648-655). In a similar way, the present inventors achieved a deletion of three Mb in 8% of ES cell clones after Cre transient expression (Step C, FIG. 2A; Table 3). The deletion was scored by the LONA assay using probes at each end of the deleted mouse sequence, as well as the loss of neo and hyg and the appearance of a PCR product through the deletion site containing the only remaining loxP site. Additionally, the deletion was confirmed by fluorescent in situ hybridization (data not shown).

The remainder of the human heavy chain variable region was added to the 3hVH allele in a series of 5 steps using the VELOCIGENE® genetic engineering method (Steps EH, FIG. 2B), and each step involves the precise insertion of up to 210 kb of human gene sequences. For each stage, the proximal end of each new BACvec was designed to overlap the more distal human sequences from the previous stage, and the distal end of each new BACvec contained the same region of distal mouse homology that was used in Stage A. BACvec from steps D, F and H contained neo selection cassettes, while those from steps E and G contained hyg selection cassettes, whereby the selections alternated between G418 and hygromycin. Targeting in step D was assayed by loss of the unique PCR product through the distal loxP site of the 3hVH Hybrid Allele. The targeting of steps E to I was analyzed by loss of the previous selection cassette. In the final stage (Stage I, FIG. 2B), the neo selection cassette flanked by Frt sites (McLeod, M. et al. (1986) Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Mol Cell Biol 6, 3357-3367), was removed by transient expression of FLPe (Buchholz, F. et al. (1998). Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16, 657-662 ). The human BACvec sequences for stages D, E and G were derived from two human precursor BACs each, while those of stages F and H were derived from single bAc. Retention of human sequences was confirmed at each step using multiple probes spanning the inserted human sequences (as described above, eg, FIG. 3A, 3B and 3C). Only those clones with normal karyotype and germline potential were carried out at each stage. Final stage ES cells were still able to contribute to the germ line after nine sequential manipulations (Table 3). Mice homozygous for each of the heavy chain alleles were viable, appeared healthy, and demonstrated an essentially wild-type humoral immune system (see Example 3).

Table 3

Immunoglobulin K Light Chain Variable Gene Locus.

The k light chain variable region was humanized in eight sequential steps by direct replacement of approximately three Mb of mouse sequence containing all Vk and Jk gene segments with approximately 0.5 Mb of human sequence containing Vk gene segments and human proximal Jk in a manner similar to that of the heavy chain (FIG. 1B; Tables 2 and 4).

The variable region of the human k light chain locus contains two nearly identical 400 kb repeats separated by an 800 kb spacer (Weichhold, GM et al. (1993) and human immunoglobulin kappa locus consists of two copies that are organized in opposite polarity, Genomics 16: 503-511). Because the repeats are so similar, almost all of the locus diversity can be reproduced in mice using the proximal repeat. Additionally, a natural human allele of the k light chain locus has been reported to lack distal repeat (Schaible, G. et al. (1993) The immunoglobulin kappa locus: polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals, Hum Genet 91: 261-267). Approximately three Mb of mouse k light chain variable gene sequence were replaced with approximately 0.5 Mb of human k light chain variable gene sequence to effectively replace all mouse Vk and Jk gene segments with the proximal human Vk and all human Jk gene segments (FIG. 2C and 2D; Tables 2 and 4). Unlike the method described in Example 1 for the heavy chain locus, the entire mouse Vk gene region, which contains all the Vk and Jk gene segments, was deleted in a three-step process before any human sequence. First, a neo cassette was inserted into the proximal end of the variable region (Step A, FIG. 2C). Next, a hyg cassette was inserted into the distal end of the k locus (Step B, FIG. 2C). The LoxP sites were repositioned within each selection cassette so that Cre treatment induced deletion of the remaining 3 Mb of the mouse Vk region along with both resistance genes (Step C, FIG. 2C).

A human genomic fragment approximately 480 kb in size containing the complete immunoglobulin k light chain variable region was inserted in four sequential steps (FIG. 2D; Tables 2 and 4), with up to 150 kb of k light chain sequence of human immunoglobulin inserted in a single step, using methods similar to those used for the heavy chain (see Example 1). The final hygromycin resistance gene was removed by transient expression of FLPe. As with the heavy chain, targeted ES cell clones were evaluated to determine the integrity of the entire human insert, the normal karyotype, and the germline potential after each step. Homozygous mice were generated for each of the k light chain alleles and were found to be healthy and normal in appearance.

Table 4

Example II

Generation of Fully Humanized Mice by Combining Multiple Humanized Immunoglobulin Alleles

At various points, ES cells carrying a portion of the human immunoglobulin heavy chain or light chain k variable repertoires as described in Example 1 were microinjected and the resulting mice mated to create multiple versions of humanized v ElOCIMMUNE mice. ® with progressively larger fractions of the human germline immunoglobulin repertoires (Table 5; FIG. 5A and 5B). Humanized VELOCIMMUNE® 1 (V1) mice possess 18 human Vh gene segments and all human Dh and Jh gene segments combined with 16 human Vk gene segments and all human Jk gene segments. Humanized VELOCIMMUNE® 2 (V2) mice and humanized VELOCIMMUNE® (V3) mice have increased variable repertoires carrying a total of 39 Vh and 30 Vk, and 80 Vh and 40 Vk, respectively. Because the genomic regions encoding the mouse VH, DH, and JH gene segments, and the Vk and Jk gene segments, have been completely replaced, the antibodies produced by any version of humanized VELOCIMMUNE® mice contain human variable regions linked to regions mouse constants. The mouse A light chain loci remain intact in all versions of humanized VELOCIMMUNE® mice and serve as a comparator for the expression efficiency of the various humanized VELOCIMMUNE® light chain loci.

Doubly homozygous mice for both immunoglobulin heavy chain and light chain k humanization were generated from a subset of the alleles described in Example 1. All genotypes observed during the course of reproduction to generate mice doubly Homozygotes were produced in approximately Mendelian proportions. The homozygous male progeny for each of the human heavy chain alleles showed reduced fertility. Reduced fertility was the result of loss of mouse ADAM6 activity. The mouse heavy chain variable gene locus contains two embedded functional ADAM6 genes (ADAM6a and ADAM6b). During humanization of the mouse heavy chain variable gene locus, the inserted human genomic sequence contained an ADAM6 pseudogene. Mouse ADAM6 may be required for fertility, and therefore the lack of mouse ADAM6 genes at humanized heavy chain variable gene loci could lead to reduced fertility in these mice despite the presence of the human pseudogene. . Examples 7-9 describe the precise replacement of deleted mouse ADAM6 genes at a humanized heavy chain variable gene locus, and restoration of a wild-type fertility level in mice with a humanized immunoglobulin heavy chain locus.

Table 5

(continuation)

Example III

Lymphocyte Populations in Mice with Humanized Immunoglobulin Genes

The mature B lymphocyte populations in the three different versions of VELOCIMMUNE® mice were evaluated by flow cytometry.

In summary, bone marrow, spleen and thymus cell suspensions were made using conventional methods. Cells were resuspended at 5x105 cells / ml in BD Pharmingen FACS staining buffer, blocked with mouse anti-CD16 / 32 (BD Pharmingen), stained with the appropriate antibody cocktail and fixed with BD CYTOFIX ™, all of according to the manufacturer's instructions. The final cell pellets were resuspended in 0.5 ml of staining buffer and analyzed using BD FACSCALIBUR ™ and BD CELLQUEST PRO ™ software. All antibodies (BD Pharmingen) were prepared in a mass / cocktail dilution and added to a final concentration of 0.5 mg / 105 cells. The antibody cocktails for bone marrow staining (A-D) were as follows: A: anti-mouse IgMb-FITC, anti-mouse IgMa-PE, anti-mouse CD45R (B220) -APC; B: mouse anti-CD43 (S7) -PE, mouse anti-CD45R (B220) -APC; C: mouse anti-CD24 (HSA) -PE; anti-CD45R (B220) mouse-APC; D: mouse anti-BP-1-PE, mouse anti-CD45R (B220) -APC. Antibody cocktails for staining of the spleen and inguinal lymph nodes (E-H) were as follows: E: anti-mouse IgMb-FITC, anti-mouse IgMa-PE, anti-mouse CD45R (B220) -APC; F: mouse anti-Ig, light chain A1, A2, A3-FITC, anti-mouse IgK light chain-PE, mouse anti-CD45R (B220) -APC; G: mouse anti-Ly6G / C-FITC, mouse anti-CD49b (DX5) -PE, mouse anti-CD11b-APC; H: mouse anti-CD4 (L3T4) -FITC, mouse anti-CD45R (B220) -PE, mouse anti-CD8a-APC. The results are shown in FIG. 6.

Lymphocytes isolated from the spleen or lymph node from homozygous humanized VELOCIMMUNE® mice were stained for surface expression of B220 and IgM markers and analyzed using flow cytometry (FIG. 6). The sizes of the mature B220 + IgM + B lymphocyte populations in all versions of humanized VELOCIMMUNE® mice tested were practically identical to that of the wild-type mice, regardless of the number of Vh gene segments they contained. In addition, mice containing homozygous hybrid humanized immunoglobulin heavy chain loci, including those with only 3 Vh gene segments but normal mouse immunoglobulin k light chain locus or mice containing homozygous hybridized human locus k light chain locus of normal mouse immunoglobulin heavy chain, they also had normal numbers of B220 + IgM + lymphocytes in their peripheral compartments (not shown). These results indicate that chimeric loci with human variable gene segments and mouse constant regions can fully populate the mature B lymphocyte compartment. Additionally, the number of variable gene segments at the heavy chain or light chain k loci, and therefore the theoretical diversity of the antibody repertoire, does not correlate with the ability to generate populations of mature wild-type B lymphocytes. In contrast, mice with randomized fully integrated human immunoglobulin transgenes and inactivated mouse immunoglobulin loci have a low number of B lymphocytes in these compartments, and the severity of the deficit depends on the number of variable gene segments included in the transgene. (Green, LL, and Jakobovits, A. (1998) Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes, J Exp Med 188: 483-495). This demonstrates that the " in situ gene humanization" strategy results in a fundamentally different functional outcome than the randomized integrated transgenes achieved in the "more transgenic gene deletion" approach.

Allelic Exclusion and Locus Choice.

The ability to maintain allelic exclusion in heterozygous mice for different versions of the humanized immunoglobulin heavy chain locus was examined.

Humanization of the immunoglobulin loci was performed in an F1 ES line (F1H4 (Valenzuela et al., 2003)), derived from heterozygous 129S6 / SvEvTac and C57BL / 6NTac embryos. Variable gene sequences of the human heavy chain germline target the 129S6 allele, which carries the IgMa haplotype, while the unmodified mouse C576BL / 6N allele carries the IgMb haplotype. These allelic forms of IgM can be distinguished by flow cytometry using specific antibodies for the polymorphisms found in the IgMa or IgMb alleles. As shown in FIG. 6 (bottom row), B lymphocytes identified in heterozygous mice for each version of the humanized heavy chain locus express only a single allele, either IgMa (the humanized allele) or IgMb (the wild-type allele). This demonstrates that the mechanisms involved in allelic exclusion are intact in humanized VELOCIMMUNE® mice. Furthermore, the relative number of humanized allele-positive B lymphocytes (IgMa) is approximately proportional to the number of Vh gene segments present. The humanized immunoglobulin locus is expressed in approximately 30% of B lymphocytes in humanized VELOCIMMUNE® mice. 1 heterozygous, having 18 human Vh gene segments, and 50 % of B lymphocytes in humanized VELOCIMMUNE® 2 mice and 3 heterozygotes (not shown), with 39 and 80 human Vh gene segments, respectively. Remarkably, the ratio of cells expressing the humanized versus wild-type mouse allele (0.5 for humanized VELOCIMMUNE® 1 mice and 0.9 for humanized VELOCIMMUNE® 2 mice) is greater than the ratio of the number of Variable gene segments contained in humanized versus wild-type loci (0.2 for humanized VELOCIMMUNE® 1 mice and 0.4 for humanized VELOCIMMUNE® 2 mice). This may indicate that the probability of allele choice is intermediate between a random choice of one or the other chromosome and a random choice of any particular segment V RSS (recombination signal sequence). Additionally, there may be a fraction of B lymphocytes, but not all, where one allele becomes accessible for recombination, completes the process, and closes the recombination before the other allele becomes accessible. Furthermore, the uniform distribution of cells bearing surface IgM (slgM) derived from the hybrid humanized heavy chain locus or the wild type mouse heavy chain locus is evidence that the hybrid locus is operating at a normal level. In contrast, randomly integrated human immunoglobulin transgenes compete weakly with wild-type mouse immunoglobulin loci (Bruggemann, M., et al. (1989) A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. PnAS 86, 6709-6713; Green et al. (1994); Tuaillon, N. et al. (1993) Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts, Proc Natl Acad Sci USA 90: 3720-3724). This further demonstrates that the immunoglobulins produced by humanized VELOCIMMUNE® mice are functionally different from those produced by randomly integrated transgenes in mice produced by "more transgenic gene deletion" approaches.

Polymorphisms of the Ck regions are not available in 129S6 or C57BL / 6N to examine allelic exclusion of humanized versus non-humanized k light chain loci. However, all humanized VELOCIMMUNE® mice possess wild-type mouse A-chain locus, therefore it is possible to see whether rearrangement and expression of humanized k-light-chain loci can prevent A-chain light expression. mouse. The ratio of the number of cells expressing the humanized k light chain to the number of cells expressing the mouse A light chain did not change relatively in the humanized VELOCIMMUNE® mice compared to the wild-type mice, regardless of the number of segments human Vk genes inserted into the k light chain locus (FIG. 6, third row from above). Furthermore, there was no increase in the number of double positive cells (k plus A), indicating that productive recombination at hybrid k light chain loci results in appropriate suppression of recombination of light chain loci. A mouse. In contrast, mice containing k light chain transgenes randomly integrated with inactivated mouse k light chain loci, but with wild-type mouse A light chain loci exhibit dramatically increased A / k ratios (Jakobovits, 1998), implying that the introduced k light chain transgenes do not work well in such mice. This further demonstrates the different functional results observed in immunoglobulins produced by humanized VELOCIMMUNE® mice compared to those produced by "more transgenic nulligenic" mice.

Lymphocyte development B.

Because the populations of mature B lymphocytes in humanized VELOCIMMUNE® mice resemble those of wild-type mice (described above), it is possible that defects in early differentiation of B lymphocytes are compensated for by the expansion of populations of mature B lymphocytes. The various stages of B lymphocyte differentiation were examined by analysis of B lymphocyte populations using flow cytometry. Table 6 sets forth the proportion of the cell fraction in each FAC-defined B lymphocyte lineage, using specific cell surface markers, in humanized VELOCIMMUNE® mice compared to wild-type littermates.

The early development of B lymphocytes occurs in the bone marrow, and the different stages of B lymphocyte differentiation are characterized by changes in the types and amounts of expression of cell surface markers. These differences in surface expression correlate with the molecular changes that occur at the immunoglobulin loci within the cell. The transition from pro-B to pre-B lymphocytes requires successful rearrangement and expression of functional heavy chain protein, while transition from pre-B stage to mature B stage is governed by correct rearrangement and expression of a ko A light chain. Thus, the ineffective transition between stages of B lymphocyte differentiation can be detected by changes in the relative populations of B lymphocytes at a given stage.

Table 6

(continuation)

No major defects in B lymphocyte differentiation were observed in any of the humanized VELOCIMMUNE® mice. The introduction of human heavy chain gene segments does not appear to affect the pro-B to pre-B transition, and the introduction of human k light chain gene segments does not affect the transition from pre-B to B in humanized VELOCIMMUNE® mice. . This demonstrates that "reverse chimeric" immunoglobulin molecules possessing human variable regions and mouse constants function normally in the context of B-cell signaling and co-receptor molecules leading to appropriate B-cell differentiation in an environment of mouse. In contrast, the balance between different populations during B lymphocyte differentiation is altered to varying degrees in mice that contain randomly integrated immunoglobulin transgenes and inactivated endogenous heavy chain or light chain k (Green and Jakobovits (1998)).

Example IV

Repertoire of Variable Genes in Mice with Humanized Immunoglobulins

The use of human variable gene segments in the humanized antibody repertoire of humanized VELOCIMMUNE® mice was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) of human variable regions from multiple sources, including splenocytes and hybridoma cells . Variable region sequence, gene segment usage, somatic hypermutation, and binding diversity of rearranged variable region gene segments were determined.

In summary, total RNA was extracted from 1 x 107-2 x 107 splenocytes or approximately 104-105 hybridoma cells using TRIZOL ™ (Invitrogen) or the Qiagen RNEASY ™ Mini Kit (Qiagen) and primed with constant region specific primers mouse using the SUPERSCRIPT ™ III one-step RT-PCR system (Invitrogen). Reactions were carried out with 2-5 µl of RNA from each sample using the aforementioned 3 'constant specific primers paired with pooled leader primers for each family of human variable regions for both the heavy and light chain k, by separated. Reagent and primer volumes and RT-PCR / PCR conditions were performed according to the manufacturer's instructions. Primer sequences were based on multiple sources (Wang, X. and Stollar, BD (2000) Human immunoglobulin variable region gene analysis by single cell RT-PCR, J Immunol Methods 244: 217-225; Ig-primer sets, Novagen ). When appropriate, nested secondary PCR reactions were carried out with pooled family-specific conserved framework region primers and the same 3 'mouse immunoglobulin constant specific primer used in the primary reaction. Aliquots (5 pl) of each reaction were analyzed by agarose electrophoresis and the reaction products were purified from agarose using a MONTAGE ™ gel extraction kit (Millipore). The purified products were cloned using the TOPO ™ TA cloning system (Invitrogen) and transformed into E. coli DH10p cells by electroporation. Individual clones from each transformation reaction were selected and grown in 2 ml LB broth cultures with antibiotic selection overnight at 37 ° C. Plasmid DNA was purified from bacterial cultures using a kit-based approach (Qiagen).

Use of Variable Immunoglobulin Genes.

Plasmid DNA from both the heavy and light chain k clones were sequenced with T7 or M13 reverse primers on the ABI 3100 Genetic Analyzer (Applied Biosystems). The raw sequence data was imported into SEQUENCHER ™ (v4.5, Gene Codes). Each sequence was assembled into contigs and aligned with human immunoglobulin sequences using the IMGT V-Quest search function (Brochet, X. et al. (2008) IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis. Nucleic Acids Res 36: W503-508) to identify the use of human segments Vh, Dh, Jh and Vk, Jk. The sequences were compared with the germline sequences for analysis of somatic hypermutation and recombination binding.

Mice were generated from ES cells containing the initial heavy chain modification (3hVH-CRE hybrid allele, bottom of Figure 2A) by RAG supplementation (Chen, J. et al. (1993) RAG-2- deficient blastocyst complementation: an assay of gene function in lymphocyte development, Proc Natl Acad Sci USA 90: 4528-4532) and the cDNA was prepared from splenocyte RNA. The cDNA was amplified using primer sets (described above) specific for the predicted chimeric heavy chain mRNA that would arise by V (D) J recombination within inserted human gene segments and subsequent splicing to domains mouse IgM or IgG constants. Sequences derived from these cDNA clones (not shown) demonstrated that adequate V (D) J recombination had occurred within human variable gene sequences, that rearranged human V (D) J gene segments were cut and spliced appropriately under constant mouse domains and that class change recombination had occurred. Further sequence analysis of mRNA products from subsequent hybrid immunoglobulin loci was performed.

In a similar experiment, B lymphocytes from wild-type unimmunized mice and humanized VELOCIMMUNE® were separated by flow cytometry based on the surface expression of B220 and IgM or IgG. B220 + IgM + IgG + or (SLGG +) cells were pooled surface and V ^h sequences and Vk were obtained after amplification by RT-PCR and cloning (described above). Representative use of genes was recorded in a set of RT-PCR amplified cDNAs from humanized VELOCIMMUNE® 1 mice (Table 7) and humanized VELOCIMMUNE® 3 mice (Table 8) without immunization (* defective RSS; f absent or pseudogene).

Table 7

V ^h Observed D ^h Observed V k Observed

Jk Observada

Jk Observed

J ^h Observed

Table 8

V ^h Observed D ^h Observed Vk Observed

(continuation)

As shown in Tables 7 and 8, almost all functional human V ^h , D ^h , J ^h , Vk, and Jk gene segments are used. Of the functional variable gene segments described but not detected in the humanized VELOCIMMUNE® mice from this experiment, several have been reported to have defective recombination signal sequences (RSS) and are therefore not expected to be expressed (Feeney , AJ. (2000) Factors that influence formation of B cell repertoire. Immunol Res 21: 195-202). Analysis of several different sets of immunoglobulin sequences from several humanized VELOCIMMUNE® mice, isolated from both untreated and immunized repertoires, has shown the use of these gene segments, albeit at lower frequencies (data not shown). Aggregated gene usage data has shown that all functional human VH, DH, JH, Vk and Jk gene segments contained in humanized VELOCIMMUNE® mice have been observed in several untreated and immunized repertoires (data not shown). Although the human VH7-81 gene segment has been identified in the analysis of human heavy chain locus sequences (Matsuda, F. et al. (1998) The complete nucleotide sequence of the human immunoglobulin heavy chain variable locus region, J Exp Med 188: 2151-2162), is not present in humanized VELOCIMMUNE® mice as confirmed by resequencing of the entire humanized VELOCIMMUNE® 3 mouse genome.

Antibody heavy and light chain sequences are known to show exceptional variability, especially in short polypeptide segments within the rearranged variable domain. These regions, known as hypervariable regions or complementarity determining regions (CDRs) create the binding site for the antigen in the structure of the antibody molecule. The intermediate polypeptide sequences are called conserved framework regions (FR). There are three CDRs (CDR1, CDR2, CDR3) and 4 FRs (FR1, FR2, FR3, FR4) in both heavy and light chains. A CDR, CDR3, is unique in that this CDR is created by recombining both the V ^h , D ^h, and J ^h and Vk and Jk gene segments and generates a significant amount of repertoire diversity before antigen is found. This binding is imprecise due to both nucleotide deletions through exonuclease activity and non-template encoded additions through terminal deoxynucleotidyl transferase (TdT), and thus allows new sequences to emerge from the recombination. Although FRs can show substantial somatic mutation due to the high mutability of the variable region as a whole, the variability, however, is not evenly distributed across the variable region. CDRs are concentrated and localized regions of high variability on the surface of the antibody molecule that allow binding to the antigen. Heavy chain and light chain sequences of antibodies selected from humanized VELOCIMMUNE® mice around the CDR3 junction demonstrating binding diversity are shown in FIG. 7A and 7B, respectively.

As shown in FIG: 7A, additions of non-template coded nucleotides (N additions) are observed at the V ^h -D ^h and D ^h -J ^h junction in antibodies from humanized VELOCIMMUNE® mice, indicating adequate function of TdT with human segments. Endpoints of the segments V ^h, D ^h and J ^h relative to their germline counterparts indicate that there has also been exonuclease activity. Unlike the heavy chain locus, rearrangements of the human k light chain show little or no addition of TdT to CDR3, which is formed by recombination of the Vk and Jk segments (FIG. 7B). This is expected due to the lack of TdT expression in mice during light chain rearrangements in the transition from preB to B lymphocytes. The observed diversity in CDR3 of rearranged human Vk regions is introduced predominantly through exonuclease activity. during the recombination event.

Somatic hypermutation.

Additional diversity is added to the variable regions of the rearranged immunoglobulin genes during the germ center reaction by a process called somatic hypermutation. B lymphocytes expressing somatically mutated variable regions compete with other B lymphocytes for access to antigen presented by follicular dendritic cells. Those B lymphocytes with higher affinity for the antigen will expand further and undergo a class change before leaving for the periphery. Therefore, B lymphocytes expressing changed isotypes have generally found antigen and experienced germ center reactions and will have a higher number of mutations relative to untreated B lymphocytes. Additionally, the predominantly untreated slgM + B lymphocyte variable region sequences would be expected to have relatively fewer mutations than the slgG + B lymphocyte variable sequences that have undergone selection by antigen.

Sequences of random V ^h or Vk clones of slgM + or slgG + B lymphocytes from unimmunized humanized VELOCIMMUNE® mice or slgG + B lymphocytes from immunized mice were compared to their germline variable gene segments and the changes relative to the germline sequence. The resulting nucleotide sequences were translated in silico and mutations leading to amino acid changes were also noted. Data was collected from all variable regions and the percent change at a given position was calculated (FIG. 8).

As shown in FIG. 8, Human heavy chain variable regions derived from slgG + B lymphocytes from unimmunized humanized VELOCIMMUNE® mice show much more nucleotides relative to slgM + B lymphocytes from the same groups of splenocytes, and Heavy chain variable regions derived from immunized mice. show even more changes. The number of changes increases in Complementarity Determining Regions (CDRs) relative to conserved framework regions, indicating selection by antigen. The corresponding amino acid sequences of the human heavy chain variable regions also show a significantly higher number of mutations in IgG versus IgM and even more in immunized IgG. These mutations again appear to be more frequent in CDRs compared to conserved framework sequences, suggesting that antibodies were screened with antigen in vivo. A similar increase in the number of nucleotide and amino acid mutations is observed in Vk sequences derived from IgG + B lymphocytes from immunized mice.

The use of genes and somatic hypermutation observed in humanized VELOCIMMUNE® mice demonstrate that essentially all of the gene segments present are capable of rearranging to form fully functional reverse chimeric antibodies in these mice. Additionally, antibodies derived from humanized VELOCIMMUNE® mice fully participate in the mouse immune system to undergo selection and affinity maturation to create fully mature human antibodies that can effectively neutralize your target antigen. Humanized VELOCIMMUNE® mice can generate strong immune responses to multiple classes of antigens resulting in the use of a wide range of human antibodies that are both high affinity and suitable for therapeutic use (data not shown).

Example V

Analysis of the Lymphoid Structure and Serum Isotypes

Macroscopic structures of the spleen, inguinal lymph nodes, Peyer's patches, and thymus from tissue samples from humanized wild-type or VELOCIMMUNE® mice stained with H&E (hematoxylin and eosin) were examined by light microscopy. Serum immunoglobulin isotype levels collected from wild-type mice and humanized VELOCIMMUNE® were analyzed using LUMINEX ™ technology.

Structure of the Lymphoid Organs.

The structure and function of lymphoid tissues depend in part on the proper development of hematopoietic cells. A defect in the development or function of B lymphocytes can show up as an alteration in the structure of lymphoid tissues. Upon analysis of the stained tissue sections, no significant differences in the appearance of the secondary lymphoid organs were identified between wild type mice and humanized VELOCIMMUNE® (data not shown).

Serum Immunoglobulin Levels.

The expression level of each isotype is similar in wild type mice and humanized VELOCIMMUNE® (FIG.

9A, 9B and 9C). This demonstrates that the humanization of the variable gene segments had no apparent adverse effect on class change or immunoglobulin expression and secretion, and therefore appears to maintain all necessary endogenous mouse sequences for these functions.

Example VI

Immunization and Antibody Production in Mice with Humanized Immunoglobulins

Different versions of humanized VELOCIMMUNE® mice were immunized with antigen to examine the humoral response to exposure with foreign antigen.

Immunization and Development of Hybridoma.

Humanized and wild-type VELOCIMMUNE® mice can be immunized with an antigen in the form of protein, DNA, a combination of DNA and protein, or cells expressing the antigen. Animals are generally boosted every three weeks for a total of two to three times. After each antigen boost, serum samples are collected from each animal and analyzed for antigen-specific antibody responses by determination of serum titer. Before fusion, mice received a final boost prior to fusion of 5 µg protein or DNA, as desired, by intraperitoneal and / or intravenous injections. Splenocytes are collected and fused to Ag8.653 myeloma cells in an electrofusion chamber according to the protocol suggested by the manufacturer (Cyto Pulse Sciences Inc., Glen Bumie, MD). Ten days after culturing, hybridomas are screened for antigen specificity using an ELISA assay (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. "Cold Spring Harbor Press, New York). Alternatively Antigen-specific B lymphocytes are isolated directly from immunized humanized VELOCIMMUNE® mice and screened using standard techniques, including those described herein, to obtain human antibodies specific for an antigen of interest.

Determination of Serum Titers.

To monitor the anti serum antigen response in animals, serum samples are collected approximately 10 days after each boost and titers are determined using antigen specific ELISA. Briefly, Nunc MAXISORP ™ 96-well plates are coated with 2 jg / ml antigen overnight at 4 ° C and blocked with bovine serum albumin (Sigma, St. Louis, MO). Serum samples in 3-fold serial dilutions are allowed to bind to the plates for one hour at room temperature. The plates are then washed with PBS containing 0.05% Tween-20 and bound IgG is detected using HRP-conjugated goat anti-mouse Fc (Jackson Immuno Research Laboratories, Inc., West Grove, PA) for the total IgG titer, or biotin-labeled isotype-specific or light chain polyclonal antibodies (SouthernBiotech Inc.) for isotype-specific titers, respectively. For biotin-labeled antibodies, after plate washing, HRP-conjugated streptavidin (Pierce, Rockford, IL) is added. All plates are developed using colorimetric substrates such as BD OPTEIA ™ (BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1M phosphoric acid, the optical absorptions are recorded at 450nm and the data is analyzed using Graph's PRISM ™ software Pad. The dilutions required to obtain the background signal twice are defined as a title.

In one experiment, humanized VELOCIMMUNE® mice were immunized with the human interleukin-6 receptor (hIL-6R). A representative set of serum titers for hIL-6R immunized wild-type and VELOCIMMUNE® mice is shown in FIGS. 10A and 10B.

Wild type humanized VELOCIMMUNE® mice mounted strong responses to IL-6R with similar titer ranges (FIG. 10A). Several mice from the humanized and wild-type VELOCIMMUNE® cohorts achieved a maximum response after a single antigen boost. These results indicate that the immune response strength and kinetics of this antigen were similar in humanized and wild type VELOCIMMUNE® mice. These antigen-specific antibody responses were further analyzed to examine the particular isotypes of the antigen-specific antibodies found in the sera. Both humanized and wild-type VELOCIMMUNE® groups predominantly elicited a response to IgG1 (FIG. 10B), suggesting that the class change during the humoral response is similar in mice of each type.

Determination of the Affinity of Antibody to Antigen in Solution.

An ELISA-based solution competition assay is generally designed to determine the antigen-binding affinity of the antibody.

In summary, the antibodies in conditioned medium are pre-mixed with serial dilutions of antigenic protein ranging from 0 to 10 mg / ml. Antibody-antigen mixture solutions are then incubated for two to four hours at room temperature to achieve binding equilibria. The amounts of free antibody in the mixtures are then measured using a quantitative sandwich ELISA. The ninety-six well MAXISORB ™ plates (VWR, West Chester, PA) are coated with 1 pg / ml antigenic protein in PBS solution overnight at 4 ° C followed by nonspecific blocking with BSA. Solutions of the antigen antibody mixture are transferred to these plates followed by a one hour incubation. The plates are then washed with wash buffer and the antibodies bound to the plate were detected with a HRP-conjugated goat anti-mouse IgG polyclonal antibody reagent (Jackson Immuno Research Lab) and grown using colorimetric substrates such as BD OPTEIA ™ (BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1M phosphoric acid, the optical absorptions are recorded at 450nm and the data is analyzed using the PRISM ™ software from Graph Pad. The dependence of the signals on the antigen concentrations in solution is analyzed with a 4-parameter fit analysis and reported as IC50, the antigen concentration required to achieve a 50% reduction in the signal of the antibody samples without the presence of antigen in solution.

In one experiment, humanized VELOCIMMUNE® mice were immunized with hIL-6R (as described above). FIG. 11A and 11B show a representative set of affinity measurements for anti-hIL6R antibodies from humanized and wild-type VELOCIMMUNE® mice.

After immunized mice receive a third antigen boost, serum titers are determined by ELISA. Splenocytes are isolated from cohorts of selected humanized wild type and VELOCIMMUNE® mice and fused with Ag8.653 myeloma cells to form hybridomas and cultured under selection (as described above). Of a total of 671 anti-IL-6R hybridomas produced, 236 were found to express antigen-specific antibodies. The medium collected from the antigen positive wells was used to determine the affinity of the antigen-binding antibody using a competition ELISA in solution. Antibodies derived from humanized VELOCIMMUNE® mice show a wide range of affinity for antigen binding in solution (FIG. 11A). In addition, 49 of the 236 anti-IL-6R hybridomas were found to block IL-6 binding to the receptor in an in vitro bioassay (data not shown). Additionally, these 49 anti-IL-6R blocking antibodies showed a range of high solution affinities similar to those of blocking antibodies derived from parallel immunization of wild-type mice (FIG. 11B).

Example VII

Construction of a Mouse ADAM6 Addressing Vector

A targeting vector for the insertion of mouse ADAM6a and ADAM6b genes into a humanized heavy chain locus was constructed using VELOCIGENE® Genetic Engineering Technology ( supra) to modify an Artificial Bacterial Chromosome (BAC) 929d24 obtained from Dr. Fred Alt (Havard University). BAC 929d24 DNA was engineered to contain genomic fragments containing the mouse ADAM6a and ADAM6b genes and a hygromycin cassette for targeted deletion of a human ADAM6 pseudogene (hADAM6 ^) located between the V ^h 1-2 gene segments and human V ^h 6-1 from a humanized heavy chain locus (FIG. 12).

First, a genomic fragment containing the mouse ADAM6b gene, 800 bp upstream sequence (5 ') and 4800 bp downstream sequence (3') was subcloned from the 929d24 BAC clone. A second genomic fragment containing the mouse ADAM6a gene, 300 bp upstream sequence (5 ') and 3400 bp sequence downstream (3 '), it was subcloned separately from the 929d24 BAC clone. The two genomic fragments containing the mouse ADAM6b and ADAM6a genes were linked to a hygromycin cassette flanked by Frt recombination sites to create the targeting vector (Mouse ADAM6 Targeting Vector, FIG. 20; SEQ ID NO: 3 ). Different restriction enzyme sites were engineered at the 5 'end of the targeting vector following the mouse ADAM6b gene and at the 3' end following the mouse ADAM6a gene (bottom of FIG. 12) for binding to the humanized heavy chain locus.

A separate modification was made in a BAC clone containing a replacement of the mouse heavy chain locus with the human heavy chain locus, including the human ADAM6 pseudogene located between the gene segments V ^h 1-2 and V ^h 6-1 humanized locus for posterior binding of the mouse ADAM6 targeting vector (FIG. 13).

In summary, a neomycin cassette flanked by loxP recombination sites was engineered to contain homology arms containing the human genomic sequence at positions 3 'of the human V ^h 1-2 gene segment (5' to hADAM6 ^) and 5 'of the human V ^h 6-1 gene segment (3' with respect to hADAM6 ^; see middle of FIG. 13). The location of the insertion site of this targeting construct was approximately 1.3 kb 5 'and 350 bp 3' of the human ADAM6 pseudogene. The targeting construct also included the same restriction sites as the mouse ADAM6 targeting vector to allow for subsequent BAC binding between the modified BAC clone containing the human ADAM6 pseudogene deletion and the mouse ADAM6 targeting vector.

After digestion of the BAC DNA derived from both constructs, the genomic fragments were pooled to construct an engineered BAC clone containing a humanized heavy chain locus containing an ectopically placed genomic sequence comprising nucleotide sequences ADAM6a and ADAM6b from mouse. The final targeting construct for the deletion of a human ADAM6 gene within a humanized heavy chain locus and the insertion of mouse ADAM6a and ADAM6b sequences into contained ES cells, 5 'to 3', a 5 'genomic fragment containing 13 kb of human genomic sequence 3 'of the human V ^h 1-2 gene segment, 800 bp of mouse genomic sequence downstream of the mouse ADAM6b gene, mouse ADAM6b gene, 4800 bp of genomic sequence upstream of the ADAM6b gene of mouse, a 5 'Frt site, a hygromycin cassette, a 3' Frt site, 300 bp mouse genomic sequence downstream of the mouse ADAM6a gene, mouse ADAM6a gene, 3400 bp mouse genomic sequence upstream of the mouse mouse ADAM6a gene, and a 3 'genomic fragment containing 30 kb of human genomic sequence 5' from the human V ^h 6-1 gene segment (bottom of FIG. 13).

The engineered BAC clone (described above) was used to electroporate mouse ES cells containing a humanized heavy chain locus to create modified ES cells comprising an ectopically placed mouse genomic sequence comprising mouse ADAM6a and ADAM6b sequences within a humanized heavy chain locus. Positive ES cells containing the mouse ectopic genomic fragment within the humanized heavy chain locus were identified by a quantitative PCR assay using TAQMAN ™ probes (Lie, YS and Petropoulos, CJ (1998) Advances in quantitative PCR technology: 5 nuclease assays Curr Opin Biotechnol 9 (1): 43-48). The upstream and downstream regions outside the modified portion of the humanized heavy chain locus were confirmed by PCR using primers and probes located within the modified region to confirm the presence of the mouse ectopic genomic sequence within the chain locus. humanized weighing, as well as the hygromycin cassette. The nucleotide sequence through the upstream insertion point included the following, indicating the human heavy chain genomic sequence upstream of the insertion point and an I-Ceu I restriction site (contained in parentheses below) linked contiguously to the mouse genomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGT TCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TGGGAGTACG GGGCGTTGGA AGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCT AAGGTAGCGA G) GGGATGACAG ATTCTCTGTT CAGTGCACTC AGGGTCTGCC TCCACGAGAA TCACCATGCC CTTTCTCAAG ACTGTGTTCT GTGCAGTGCC CTGTCAGTGG (SEQ ID NO: 4). The nucleotide sequence through the insertion point downstream at the 3 'end of the target region included the following, indicating the mouse genomic sequence and a Pl-Sce I restriction site (contained within the parentheses below) linked contiguously with the genomic sequence of human heavy chain downstream of the insertion point (AGGGGTCGAG GGGGAATTTT ACAAAGAACA AAGAAGCGGG CATCTGCTGA CATGAGGGCC GAAGTCAGGC TCCAGGCAGC GGGAGCTCCA CCGCGGTGGC GCCATTTCAT TACCTCTTTC TCCGCACCCG ACATAGATAAAGCTT) ATCCCCCACC AAGCAAATCC CCCTACCTGG GGCCGAGCTT CCCGTATGTG GGAAAATGAA TCCCTGAGGT CGATTGCTGC ATGCAATGAA ATTCAACTAG (SEQ ID NO: 5) .

The targeting ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® engineering method (see, eg, US Patent Nos. 7,6598,442, 7,576,259, 7,294,754). Mice bearing a humanized heavy chain locus containing a mouse ectopic genomic sequence comprising mouse ADAM6a and ADAM6b sequences were identified by genotyping using a modification of the allele assay (Valenzuela et al., 2003) that detected the presence of mouse ADAM6a and ADAM6b genes within humanized heavy chain locus.

Mice bearing a humanized heavy chain locus containing mouse ADAM6a and ADAM6b genes are reproduced with a mouse strain of FLPe suppressor (see, eg, Rodriguez, CI et al. (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre-lox P. Nature Genetics 25: 139-140) to remove any FRT-flanked hygromycin cassette introduced by the targeting vector that is not removed, for example, in the ES cell phase or in the embryo . Optionally, the hygromycin cassette is preserved in mice.

The offspring are genotyped and a heterozygous offspring is selected for a humanized heavy chain locus containing a mouse ectopic genomic fragment comprising mouse ADAM6a and ADAM6b sequences to characterize mouse ADAM6 gene expression and fertility.

Example VIII

Characterization of ADAM6 Rescue Mice

Flow cytometry.

Three 25-week-old mice homozygous for the human k light and human heavy chain (H / k) variable gene locus and three 18-20 week-old mice homozygous for the human k light and human heavy chain having the fragment ectopic mouse genomic encoding the mouse ADAM6a and ADAM6b genes within both alleles of the human heavy chain locus (H / k-A6) were sacrificed for identification and analysis of lymphocyte cell populations by FAC on the BD LSR II system (Bd Bioscience). Lymphocytes were screened for specific cell lines and analyzed for progression through various stages of B lymphocyte development. Tissues collected from animals included blood, spleen, and bone marrow. Blood was collected in BD microtainer tubes with EDTA (BD Biosciences). Bone marrow was removed from femurs by extensive washing with complete RPMI medium supplemented with fetal calf serum, sodium pyruvate, HEPES, 2-mercaptoethanol, non-essential amino acids, and gentamicin. The red blood cells from the blood, spleen and bone marrow preparations were lysed with an ammonium chloride based lysis buffer (eg ACK lysis buffer), followed by washing with complete RPMI medium.

For staining cell populations, 1 x 10 6 cells from various tissue sources were incubated with mouse anti-CD16 / CD32 (2.4G2, BD Biosciences) on ice for 10 minutes, followed by labeling with one or a combination of the following antibody cocktails for 30 min on ice.

Bone marrow: anti-FITC-CD43 (1B11, BioLegend), PE-ckit (2B8, BioLegend), PeCy7-IgM (II / 41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend) , APC-eFluor780-B220 (RA3-6B2, eBioscience), mouse A700-CD19 (1D3, BD Biosciences).

Peripheral blood and spleen: anti-FITC-K (187.1, BD Biosciences), PE-A (RML-42, BioLegend), PeCy7-IgM (II / 41, eBioscience), PerCP-Cy5.5-IgD (11-26c .2a, BioLegend), mouse APC-CD3 (145-2C11, BD), A700-CD19 (1D3, BD), mouse APC-eFluor780-B220 (RA3-6B2, eBioscience). After incubation with the labeled antibodies, the cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on an LSRII flow cytometer and analyzed with FlowJo. The results of a representative H / k mouse and H / K-A6 are shown in Figs. 14-18.

The results demonstrate that B lymphocytes from H / K-A6 mice progress through the stages of B lymphocyte development in a similar way to H / k mice in the bone marrow and peripheral compartments, and show normal patterns of maturation once they enter the periphery. H / K-A6 mice demonstrated an increase in CD43intCD19 + cell population compared to H / k mice (FIG. 16B). This may indicate accelerated IgM expression from the humanized heavy chain locus containing an ectopic mouse genomic fragment comprising the mouse ADAM6a and ADAM6b sequences in H / K-A6 mice. At the periphery, the B and T lymphocyte populations of H / K-A6 mice appear normal and similar to those of H / k mice.

Testicular Morphology and Sperm Characterization.

To determine whether infertility in mice having humanized immunoglobulin heavy chain variable loci is due to defects in the testes and / or sperm production, the morphology of the testes and the sperm content of the epididymis were examined.

Briefly, the testes of two groups of five mice per group were dissected and weighed (Group 1: mice homozygous for human K heavy and light chain variable gene loci, mADAM6 '/ _; Group 2: heterozygous mice for gene locus Human heavy chain variables and homozygotes for human K heavy and light chain variable gene loci, mADAM6 '/') with the epididymis intact. The samples were then fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin (HE) stain. Sections of the testes (2 testes per mouse, for a total of 20) were examined for defects in morphology and evidence of sperm production, while sections of the epididymis were examined for the presence of sperm.

In this experiment, no difference in testis weight or morphology was observed between mADAM6 - / - mice and mADAM6 +/- mice. Sperm were observed in all genotypes, both in the testes and in the epididymis. These results establish that the absence of mouse ADAM6a and ADAM6b genes does not lead to detectable changes in testis morphology, and that sperm is produced in mice in the presence and absence of these two genes. Therefore, fertility defects in male ADAM6 - / - mice are unlikely to be due to low sperm production.

Motility and Spermatic Migration.

Mice lacking other members of the ADAM gene family are infertile due to defects in motility or migration of sperm. Sperm migration is defined as the ability of sperm to pass from the uterus to the oviduct, and is normally required for fertilization in mice. To determine if deletion of mouse ADAM6a and ADAM6b affects this process, sperm migration was evaluated in mADAM6 - / - mice. Sperm motility was also evaluated.

Briefly, sperm from testes were obtained from (1) mice heterozygous for human heavy chain variable gene locus and homozygous for human k light chain variable gene locus (ADAM6 +/-); (2) mice homozygous for human heavy chain variable gene locus and homozygous for human k light chain variable gene locus (ADAM6 - / -); (3) mice homozygous for human heavy chain variable gene loci and homozygous for wild-type k light chain (ADAM6 - / - im); and, (4) wild type (TS) C57 BL / 6 mice. No significant abnormalities in sperm count or overall sperm motility were observed by inspection. For all mice, cluster dispersion was observed, indicating that each sperm sample was able to penetrate the cluster cells and bind to the zona pellucida in vitro. These results establish that ADAM6 - / - mice have sperm capable of penetrating the cluster and attaching to the zona pellucida.

In vitro mouse egg fertilization (IVF) was performed using mouse sperm as described above. A slightly smaller number of cleaved embryos were present for ADAM6 - / - the day after IVF, as well as a reduced number of sperm bound to the ovules. These results establish that the sperm of the ADAM6 - / - mice, once exposed to an egg, are able to penetrate the cluster and bind to the zona pellucida.

In another experiment, the ability of sperm from ADAM6 - / - mice to migrate from the uterus and through the oviduct was determined in a sperm migration assay.

In summary, a first group of five superovulated female mice was established with five ADAM6 - / - males. A second group of five superovulated female mice with five ADAM6 +/- males was established. The mating pairs were observed for copulation, and five to six hours after copulation, the uterus and attached oviduct were removed from all females and washed extensively for analysis. The wash solutions were checked for eggs to verify ovulation and obtain a sperm count. Sperm migration was evaluated in two different ways. First, both oviducts were removed from the uterus, flushed with saline, and any identified sperm were counted. The presence of ovules was also observed as evidence of ovulation. Second, the oviducts were left attached to the uterus and both tissues were fixed, paraffin-embedded, sectioned, and stained (as described above). Sections were examined for the presence of sperm, both in the uterus and in both oviducts.

For the five females paired with the five ADAM6 - / - males, very few sperm were found in the oviduct wash solution. The washing solutions of the oviducts of the five females paired with the five ADAM6 +/- males showed a sperm level of 25 to 30 times higher (average, n = 10 oviducts) than that present in the washing solutions of the oviducts of the five females paired with the five ADAM6 males - / -.

Histological sections of the uterus and oviduct were prepared. Sections were examined for the presence of sperm in the uterus and oviduct (colliculus tubarius). Inspection of the histological sections of the oviduct and uterus revealed that in female mice paired with ADAM6 - / - mice, sperm was found in the uterus but not in the oviduct. Additionally, sections of females paired with ADAM6 - / - mice revealed that no sperm were found at the uterotubal junction (UTJ). In sections of females paired with ADAM6 +/- mice, sperm were identified in the UTJ and in the oviduct.

These results establish that mice that lack the ADAM6a and ADAM6b genes produce sperm that show a migration defect in vivo . In all cases, sperm were observed within the uterus, indicating that copulation and sperm release apparently occur normally, but few or no sperm were observed within the oviducts after intercourse, as measured by sperm count. or histological observation. These results establish that mice lacking the ADAM6a and ADAM6b genes produce sperm that exhibit an inability to migrate. from the uterus to the oviduct. This defect appears to lead to infertility because sperm cannot cross the uterotubal junction into the oviduct, where the eggs are fertilized. Taken together, all of these results converge to support the hypothesis that mouse ADAM6 genes help direct normally motile sperm to migrate out of the uterus, through the uterotubal junction and the oviduct, and thus approach an egg to achieve the fertilization event. The mechanism by which ADAM6 achieves this may be directly by action of the ADAM6 proteins, or by coordinated expression with other proteins, eg, other ADAM proteins, in the sperm, as described below.

Expression of the ADAM Gene Family.

An ADAM protein complex is known to be present as a complex on the surface of maturing sperm. Mice lacking other members of the ADAM gene family lose this complex as sperm mature and show a reduction of multiple ADAM proteins in mature sperm. To determine whether the lack of ADAM6a and ADAM6b genes affects other ADAM proteins in a similar way, Western blots of protein extracts from testis (immature sperm) and epididymis (mature sperm) were analyzed to determine the expression levels of other members of the ADAM gene family.

In this experiment, protein extracts from four ADAM6 - / - and four ADAM6 +/- mice were analyzed. The results showed that the expression of ADAM2 and ADAM3 was not affected in the testicle extracts. However, ADAM2 and ADAM3 were dramatically reduced in epididymal extracts. This demonstrates that the absence of ADAM6a and ADAM6b in sperm from ADAM6 - / - mice can have a direct effect on the expression and perhaps on the function of other ADAM proteins as sperm mature (eg ADAM2 and ADAM3) . This suggests that ADAM6a and ADAM6b are part of a complex of ADAM proteins on the surface of sperm, which could be critical for proper sperm migration.

Example IX

Use of Variable Human Heavy Chain Genes in ADAM6 Rescue Mice

Use of selected human heavy chain variable genes was determined for mice homozygous for the human k heavy and light chain variable gene loci that lack the mouse ADAM6a and ADAM6b genes (mADAM6 - / -) or contain a fragment. Ectopic genomic encoding the mouse ADAM6a and ADAM6b genes (ADAM6 + / +; see Example 1) by a quantitative PCR assay using TAQMAn ™ probes (as described above).

Briefly, CD19 + B lymphocytes were purified from the spleen of mADAM6 - / - and ADAM6 + / + mice using mouse CD19 microbeads (Miltenyi Biotec) and total RNA was purified using the RNEASY ™ Mini Kit (Qiagen). Genomic RNA was removed using DNase-free RNase column treatment (Qiagen). Approximately 200 ng of mRNA was reverse transcribed into cDNA using the First Stand cDNA Synthesis Kit (Invitrogen) and then amplified with the TAQMAN ™ Universal PCR Master Mix (Applied Biosystems) using the ABI 7900 Sequence Detection System (Applied Biosystems). The relative expression of each gene was normalized to the mouse constant k (mCK). Table 9 sets forth the sense / antisense / TAQMAN ™ MGB probe combinations used in this experiment.

Table 9

(continuation)

In this experiment, the expression of the four human V ^h genes was observed in the samples analyzed. Additionally, expression levels were comparable between mADAM6 - / - and ADAM6 + / + mice. These results demonstrate that the human V ^h genes that were distal to the modification site (V ^h 3-23 and V ^h 1-69) and proximal to the modification site (V ^h 1-2 and V ^h 6-1) were capable to recombine to form a functionally expressed human heavy chain. These results demonstrate that the ectopic genomic fragment comprising the mouse ADAM6a and ADAM6b sequences inserted into a human heavy chain genomic sequence did not affect the V (D) J recombination of human heavy chain gene segments within the locus, and these mice are capable of recombining human heavy chain gene segments in a normal manner to produce immunoglobulin heavy chain functional proteins.

Example X

Identification of Variable Human Heavy Chain Regions Associated with Selected Human Light Chain Variable Regions

An in vitro expression system was constructed to determine whether a single rearranged human germline light chain could be co-expressed with human heavy chains from antigen-specific human antibodies.

Methods for generating human antibodies in genetically modified mice are known (see, eg, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE® humanized mouse). Humanized VELOCIMMUNE® mouse technology involves the generation of a genetically modified mouse having a genome comprising human light and heavy chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibodies produced from a humanized VELOCIMMUNE® mouse is completely human. Initially, the high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As described below, antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate a fully human antibody that contains a non-IgM isotype, eg, wild-type or modified IgG1, IgG2, IgG3 or IgG4. Although the selected constant region may vary according to specific use, the high affinity antigen binding characteristics and target specificity reside in the variable region.

A humanized VELOCIMMUNE® mouse was immunized with an angiogenesis promoting growth factor (Antigen C) and antigen specific human antibodies were isolated and sequenced for use of the V gene using standard techniques recognized in the art. Selected antibodies were cloned into human heavy and light chain constant regions and 69 heavy chains were selected to pair with one of three human light chains: (1) the cognate k light chain attached to a human k constant region, (2) a Vk1-39Jk5 of the rearranged human germline attached to a human k constant region, or (3) a rearranged human germline Vk3-20Jk1 attached to a human k constant region. Each pair of heavy chain and light chain was cotransfected into CHO-K1 cells using standard techniques. The presence of antibodies in the supernatant was detected by anti-human IgG in an ELISA assay. The antibody titer (ng / ml) was determined for each heavy chain / light chain pair and the titers with the different rearranged germ line light chains were compared with the titers obtained with the precursor antibody molecule (i.e. heavy paired with related light chain) and percentage of natural titer was calculated (Table 10). V ^h : Heavy chain variable gene. ND: expression not detected under current experimental conditions.

______________________________ Table 10______________________________

(continuation)

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In a similar experiment, humanized VELOCIMMUNE® mice were immunized with several different antigens and the ability of selected antigen-specific human antibody heavy chains to pair with different rearranged human germline light chains (as described above) was evaluated. Antigens used in this experiment included an enzyme involved in cholesterol homeostasis (Antigen A), a serum hormone involved in the regulation of glucose homeostasis (Antigen B), a growth factor that promotes angiogenesis (Antigen C) and a cell surface receptor (Antigen D). Antigen specific antibodies were isolated from mice of each immunization group and the heavy and light chain variable regions were cloned and sequenced. From the sequence of heavy and light chains, the use of the V gene was determined and the selected heavy chains were paired with their cognate light chain or a rearranged human germline Vk1-39Jk5 region. Each heavy / light chain pair was cotransfected into CHO-K1 cells and the presence of antibody in the supernatant was detected by anti-human IgG in an ELISA assay. The antibody titer (pg / ml) was determined for each heavy chain / light chain pairing and the titers with the different rearranged human germline light chains were compared with the titers obtained with the precursor antibody molecule (i.e. heavy chain paired with cognate light chain) and percent of natural titer was calculated (Table 11). V ^h : Heavy chain variable gene. Vk: light chain variable gene k. ND: expression not detected under current experimental conditions.

Table 11

(continuation)

The results obtained from these experiments demonstrate that high affinity heavy chains with somatic mutations from different gene families can be paired with the rearranged Vk1-39Jk5 and Vk3-20Jk1 regions of the human germline and secreted from the cell as an antibody molecule. normal. As shown in Table 10, the antibody titer increased in approximately 61% (42 of 69) of the heavy chains when paired with the rearranged human Vk1-39Jk5 light chain and approximately 29% (20 of 69 ) of the heavy chains when paired with the rearranged human Vk3-20Jk1 light chain compared to the cognate light chain of the parent antibody. For approximately 20% (14 out of 69) of the heavy chains, both rearranged human germline light chains confer an increase in expression compared to the cognate light chain of the parent antibody. As shown in Table 11, the rearranged human germline Vk1-39Jk5 region conferred an increase in the expression of several specific heavy chains for a range of different classes of antigens compared to the cognate light chain for precursor antibodies. . The antibody titer increased more than twice in approximately 35% (15/43) of the heavy chains compared to the cognate light chain of the precursor antibodies. For two heavy chains (315 and 316), the increase was greater than ten-fold compared to the parent antibody. Within all heavy chains that showed increased expression relative to the cognate light chain of the parent antibody, heavy chains of family three (V ^h 3) are over-represented compared to other families of variable region genes heavy chain. This demonstrates a favorable relationship of the human V ^h 3 heavy chains to pair with the rearranged human germline Vk1-39Jk5 and Vk3-20Jk1 light chains.

Example XI

Generation of a Light Chain Locus of the Reordered Human Germ Line

Several rearranged human germline light chain targeting vectors were produced using VELOCIGENE® genetic engineering technology (see, for example, US Patent No. 6,586,251 and Valenzuela et al. (2003 ) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21 (6): 652-659) to modify clones 302 g12 and 254m04 of the Mouse Genomic Bacterial Artificial Chromosome (BAC) (Invitrogen) . Using these two BAC clones, the genomic constructs were engineered to contain a single rearranged human germline light chain region and inserted into an endogenous k light chain locus that was previously modified to suppress the variable gene segments of k endogenous and binding.

Construction of Light Chain Addressing Vectors of the Germina1Humana Reordered Line.

Three different rearranged human germ line light chain regions were made using standard art-recognized molecular biology techniques. Human variable gene segments used to construct these three regions included the rearranged human Vk1-39Jk5 sequence, a rearranged human Vk3-20Jk1 sequence, and a rearranged VpreBJA5 sequence.

A DNA segment containing exon 1 (encoding the leader peptide) and intron 1 of the mouse Vk3-7 gene was made by de novo DNA synthesis (Integrated DNA Technologies). Part of the 5 'untranslated region to a naturally occurring Blpl restriction enzyme site was included. Exons of the human Vk1-39 and Vk3-20 genes were amplified by PCR from human genomic BAC libraries. Direct primers had a 5 'extension containing the splice acceptor site of intron 1 of the mouse Vk3-7 gene. The reverse primer used for PCR of the human Vk1-39 sequence included an extension encoding human Jk5, whereas the reverse primer used for PCR of the human Vk3-20 sequence included an extension encoding human Jk1. The human VpreBJA, 5 sequence was performed by de novo DNA synthesis (Integrated DNA Technologies). A portion of the human Jk-Ck intron that includes the splice cleavage donor site was amplified by PCR from plasmid pBS-296-HA18-PISceI. The forward PCR primer included a part that encodes the extension of a human Jk5, Jk1 or JA5 sequence. The reverse primer included a Pl-Scel site, which was previously engineered into the intron.

The mouse Vk3-7 exon1 / intron 1, human variable light chain exons, and human Jk-Ck intron fragments were coupled together by overlapping extension PCR, digested with Blpl and Pl-Scel, and ligated into plasmid pBS-296-HA18-PIScel, which contained the human Vk3-15 variable gene segment promoter. A lox hygromycin cassette within plasmid pBS-296-HA18-PIScel was replaced with a FRT hygromycin cassette flanked by the Notl and Ascl sites. The Notl / PI-Scel fragment of this plasmid was linked to modified mouse BAC 254m04, which contained part of the mouse Jk-Ck intron, mouse Ck exon and approximately 75 kb of genomic sequence downstream of the mouse k locus. provided a 3 'homology arm for homologous recombination in mouse ES cells. The Notl / Ascl fragment of this BAC was then linked to modified mouse BAC 302 g12, which contained a flanked neomycin FRT cassette and approximately 23 kb of genomic sequence upstream of the endogenous k locus for homologous recombination in mouse ES cells.

Vk1-39Jk5 Addressing Vector of the Reordered Human Germ Line (FIG. 19).

Restriction enzyme sites were introduced at the 5 'and 3' ends of an engineered light chain insert to clone into a targeting vector: an Ascl site at the 5 'end and a Pl-Scel site at the 3 end. '. Within the 5'Ascl site and the 3 'Pl-Scel site, the 5' to 3 'targeting construct included a 5' homology arm containing the 5 'sequence to the endogenous mouse k light chain locus obtained from the clone. 302 g12 mouse BAC, a flanked FRT neomycin resistance gene, a genomic sequence including the human Vk3-15 promoter, a leader sequence of the mouse Vk3-7 variable gene segment, an intronic sequence of the Vk3 variable gene segment -7 mouse, an open reading frame of a rearranged human germline Vk1-39Jk5 region, a genomic sequence containing a portion of the human Jk-Ck intron, and a 3 'homology arm containing the 3' sequence of the endogenous mouse Jk5 gene segment obtained from the mouse BAC 254m04 clone (Figure 19, center). Genes and / or sequences upstream of the endogenous mouse k light chain locus and downstream of the Jk plus 3 'gene segment (eg, the endogenous 3' enhancer) were not modified by the targeting construct ( see Figure 19). The sequence of the engineered human Vk1-39Jk5 locus is shown in SEQ ID NO: 59.

The targeted insertion of the rearranged human germline Vk1-39Jk5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located in sequences within the light chain region of the rearranged human germline . In summary, the 3 'intronic sequence to the mouse Vk3-7 leader sequence was confirmed with the ULC-m1F primers (AGGTGAGGGT ACAGATAAGT GTTATGAG; SEQ lD NO: 60) and ULC-m1R (TGACAAATGC CCTAATTATAGTGATCA; SEQ lD NO: 61) . The open reading frame of the Rearranged human germline Vk1-39Jk5 region was confirmed with primers 1633-h2F (GGGCAAGTCA GAGCATTAGC A; SEQ lD NO: 62) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ lD NO: 63). The neomycin cassette was confirmed with neoF (ggtggagagg ctattcggc; SEQ lD NO: 64) and neoR (gaacacggcg gcatcag; SEQ lD NO: 65) primers. Targeted BAC DNA was then used to electroporate mouse ES cells to create modified ES cells to generate chimeric mice expressing a rearranged human germline Vk1-39Jk5 region.

Positive ES cell clones were confirmed by Taqman ™ scanning and karyotyping using probes specific for the Vk1-39Jk5 region of the engineered light chain inserted into the endogenous locus. In summary, the neoP probe (TGGGCACAAC AGACAATCGG CTG; SEQ lD NO: 66) that binds within the neomycin marker gene, ULC-m1P probe (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ lD NO: 67) that binds within the intronic sequence 3 'to the mouse Vk3-7 leader sequence, and the 1633h2P probe (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ lD NO: 68) that binds within the Vk1-39Jk5 rearranged human germline open reading frame. Then positive ES cell clones were used to implant into female mice to give rise to a litter of pups expressing the germline light chain Vk1-39Jk5 region.

Alternatively, ES cells carrying the rearranged human germline light chain Vk1-39Jk5 region are transfected with a FLP-expressing construct to remove the flanked FRT neomycin cassette introduced by the targeting construct. Optionally, the neomycin cassette is killed by crossing with mice that express FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is preserved in mice.

Addressing Vector of Vk3-20Jk1 of the Reordered Human Germ Line (FIG. 20).

Similarly, an engineered light chain locus expressing a rearranged human germline Vk3-20Jk1 region was performed using a targeting construct including, from 5 'to 3', a 5 'homology arm containing the sequence 5 'to the endogenous mouse k light chain locus obtained from the mouse BAC 302 g12 clone, a flanked FRT neomycin resistance gene, a genomic sequence including the human Vk3-15 promoter, a leader sequence of the variable gene segment Mouse Vk3-7, an intronic sequence of the mouse Vk3-7 variable gene segment, an open reading frame of a rearranged human germline Vk3-20Jk1 region, a genomic sequence containing a portion of the Jk-Ck intron human, and a 3 'homology arm containing the 3' sequence of the endogenous mouse Jk5 gene segment obtained from the mouse BAC clone 254m04 (Figure 20, center). The sequence of the engineered human Vk3-20Jk1 locus is shown in SEQ ID NO: 69.

The targeted insertion of the rearranged human germline Vk3-20Jk1 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located in sequences within the germline light chain VK3-20JKl region. rearranged human. Briefly, the intronic sequence 3 'to the mouse Vk3-7 leader sequence was confirmed with the primers ULC-m1F (SEQ ID NO: 60) and ULC-m1R (SEQ ID NO: 61). The rearranged human germline Vk3-20Jk1 region open reading frame was confirmed with primers 1635-h2F (TCCAGGCACC CTGTCTTTG; SEQ ID NO: 70) and 1635-h2R (AAGTAGCTGC TGCTAACACT CTGACT; SEQ ID n O: 71 ). The neomycin cassette was confirmed with the primers neoF (SEQ ID NO: 64) and neoR (SEQ ID NO: 65). Directed BAC DNA was then used to electroporate mouse ES cells to create modified ES cells to generate chimeric mice expressing the rearranged human germline Vk3-20Jk1 light chain.

Positive ES cell clones were confirmed by Taqman ™ scanning and karyotyping using probes specific for the Vk3-20Jk1 region of the engineered light chain inserted into the endogenous k light chain locus. In summary, neoP probe (SEQ ID NO: 66) that binds within the neomycin marker gene, ULC-m1P probe (SEQ ID NO: 67) that binds within the mouse Vk3-7 leader sequence and 1635h2P probe ( AAAGAGCCAC CCTCTCCTGC AGGG; SEQ ID NO: 72) that binds within the open reading frame of human Vk3-20Jk1. Positive ES cell clones were used to implant female mice. A litter of puppies expressing the Vk3-20Jk1 region of the light chain of the human germline.

Alternatively, ES cells carrying the human germline light chain Vk3-20Jk1 region can be transfected with a FLP-expressing construct to remove the flanked FRT neomycin cassette introduced by the targeting construct. Optionally, the neomycin cassette can be removed by crossing with mice that express FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is preserved in mice.

VpreBJA5 Addressing Vector of the Reordered Human Germ Line (FIG. 21).

Similarly, an engineered light chain locus expressing a rearranged human germline VpreBJA5 region was performed using a targeting construct including, from 5 'to 3', a 5 'homology arm containing sequence 5 'to the endogenous mouse k light chain locus obtained from the mouse BAC 302 g12 clone, a flanked FRT neomycin resistance gene, a genomic sequence including the human Vk3-15 promoter, a leader sequence of the mouse Vk3-7 variable gene segment, an intronic sequence of the mouse Vk3-7 variable gene segment, an open reading frame of a rearranged human germline VpreBJA5 region, a genomic sequence containing a portion of the human Jk-Ck intron, and a 3 'homology arm containing the 3' sequence of the endogenous mouse Jk5 gene segment obtained from mouse clone 254m04 BAC (Figure 21, center). The sequence of the engineered human VpreBJA5 locus is shown in SEQ ID NO: 73.

The targeted insertion of the rearranged human germline VpreBJA5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located in sequences within the rearranged human germline light chain VpreBJA5 region. Briefly, the intronic sequence 3 'to the mouse Vk3-7 leader sequence was confirmed with the primers ULC-m1F (SEQ ID NO: 60) and ULC-m1R (SEQ ID NO: 61). The rearranged human germline VpreBJA, 5 region open reading frame was confirmed with primers 1616-h1 F (TGTCCTCGGC CCTTGGA; SEQ ID NO: 74) and 1616-h1R (CCGATGTCAT GGTCGTTCCT; SEQ ID NO: 75) . The neomycin cassette was confirmed with the primers neoF (SEQ ID NO: 64) and neoR (SEQ ID NO: 65). Targeted BAC DNA was then used to electroporate mouse ES cells to create modified ES cells to generate chimeric mice expressing the rearranged human germline VpreBJA, 5 light chain.

Positive ES cell clones were confirmed by Taqman ™ scanning and karyotyping using probes specific for the VpreBJA, 5 region of the engineered light chain inserted into the endogenous k light chain locus. In summary, neoP probe (SEQ ID NO: 66) that binds within the neomycin marker gene, ULC-m1P probe (SEQ ID NO: 67) that binds within the mouse IgVK3-7 leader sequence, and probe 1616h1P (ACAATCCGCC TCACCTGCAC c Ct; SEQ ID NO: 76) that binds within the open reading frame of human VpreBJA, 5. Positive ES cell clones were then used to implant into female mice to give rise to a litter of pups expressing the germline light chain region.

Alternatively, ES cells carrying the rearranged human germline light chain VpreBJA, 5 region are transfected with a FLP-expressing construct to remove the flanked neomycin FRT cassette introduced by the targeting construct. Optionally, the neomycin cassette is killed by crossing with mice that express FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is preserved in mice.

Example XII

Generation of Mice expressing a single rearranged human light chain

The targeting ES cells described above were used as donor ES cells and introduced into a mouse 8 stage embryo by the VELOCIMOUSE® method (see, eg, US Patent No. 7,294. 754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor genetargeted ES cells allowing immediate phenotypic analyzes, Nature Biotech. 25 (1): 91-99. VELOCIMICE® mice are identified that independently carry a region Light chain Vk1-39Jk5, a light chain Vk3-20Jk1 region or a human germline light chain region VpreBJA5 engineered by genotyping using a modification of the allele assay (Valenzuela et al., Supra) that detects the presence of the single rearranged human germline light chain region.

The offspring are genotyped and a heterozygous or homozygous offspring for the single rearranged human germline light chain region is selected to characterize expression of the rearranged human germline light chain region.

Flow cytometry.

The expression of the rearranged human light chain region in the normal antibody repertoire of mice with common light chain was validated by analysis of immunoglobulin k and A expression in splenocytes and peripheral blood of mice with common light chain. Suspensions of harvested spleen cells and peripheral blood were made from wild type mice (n = 5), common Vk1-39Jk5 light chain heterozygotes (n = 3), common Vk1-39Jk5 light chain homozygotes (n = 3), heterozygotes. common Vk3-20Jk1 light chain (n = 2) and common Vk3-20Jk1 light chain homogeneous (n = 2) using conventional methods and stained with CD19 +, lg ^ + and lg ^ + using fluorescently labeled antibodies (BD Pharmigen ).

Briefly, 1x106 cells were incubated with mouse anti-CD16 / CD32 (clone 2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling with the following antibody cocktail for 30 minutes on ice: conjugated mouse anti-CD19 with APC (1D3 clone, BD Pharmigen), PerCP-Cy5.5 conjugated anti-mouse CD3 (clone 17A2, BioLegend), FITC-conjugated anti-mouse IgK (clone 187.1, BD Pharmigen), anti-mouse IgA conjugated with PE (clone RML-42, BioLegend). After staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on an LSRII flow cytometer and analyzed with FlowJo ™. Selection: total B lymphocytes (CD19 + CD3 '), Ig ^ B lymphocytes (Ig ^ + Ig ^' CD19 + CD3 '), IgA + B lymphocytes (IgK'IgA + CD19 + CD3'). The data collected from blood samples and splenocytes showed similar results. Table 12 shows the percentage of peripheral blood positive CD19 + B lymphocytes from a representative mouse from each group that are Ig ^ +, Ig ^ +, or Ig ^ + Ig ^ +. The percentage of CD19 + B lymphocytes in peripheral blood of wild type (TS) mice and homozygous for the common light chain Vk1-39Jk5 or Vk3-20Jk1 is shown in FIG. 22.

Table 12

Common Light Chain Expression.

The expression of each common light chain (Vk1-39Jk5 and Vk3-20Jk1) was analyzed in heterozygous and homozygous mice using a quantitative PCR assay (eg, Taqman ™).

Briefly, CD19 + B lymphocytes were purified from the spleens of wild-type mice, homozygous for a replacement of the mouse heavy chain and light chain variable region locus with k heavy chain and light chain variable region loci. Corresponding human (Hk), as well as homozygous and heterozygous mice for each rearranged human light chain region (Vk1-39Jk5 or Vk3-20Jk1) using mouse CD19 microbeads (Miltenyi Biotec) according to manufacturer specifications. Total RNA was purified from CD19 + B lymphocytes using the RNeasy ™ mini kit (Qiagen) according to the manufacturer's specifications and the genomic RNA was removed using a DNase-free RNase column treatment (Qiagen). 200 ng of mRNA was reverse transcribed into cDNA using the First Stand cDNA synthesis kit (Invitrogen) and the resulting cDNA was amplified with the Taqman ™ Universal PCR Master Mix (Applied Biosystems). All reactions were performed using the ABI 7900 Sequence Detection System (Applied Biosystems) using Taqman ™ MGB primers and probes spanning (1) the Vk-Jk junction for both common light chains, (2) the Vk gene alone (is say, Vk1-39 and Vk3-20) and (3) the mouse Ck region. Table 13 sets forth the primer and probe sequences used for this assay. Relative expression normalized to expression of the mouse Ck region. The results are shown in FIG. 23a, 23B and 23C.

Table 13

Antigen Specific Common Light Chain Antibodies.

Common light chain mice carrying a common light chain Vk1-39Jk5 or Vk3-20Jk1 at the endogenous mouse light chain k locus were immunized with p-galactosidase and antibody titer was measured.

Briefly, p-galactosidase (Sigma) was emulsified in TITERMAX ™ adjuvant (Sigma), according to the manufacturer's instructions. Wild-type mice (n = 7), Vk1-39Jk5 common light chain homozygotes (n = 2), and Vk3-20Jk1 common light chain homozygotes (n = 5) were immunized by subcutaneous injection with 100 | jg of p-galactosidase / TITERMAX ™. Mice were boosted by subcutaneous injection twice, 3 weeks apart, with 50 | jg of p-galactosidase / TITERMAX ™. After the second boost, blood was collected from anesthetized mice using retroorbital hemorrhage in serum separator tubes (BD Biosciences) according to the manufacturer's instructions. To measure IgM or IgG anti-p-galactosidase antibodies, ELISA plates (Nunc) were coated with 1 jg / ml p-galactosidase overnight at 4 ° C. Excess antigen was washed before blocking with PBS with 1% BSA for one hour at room temperature. Serial dilutions of serum were added to the plates and incubated for one hour at room temperature before washing. The plates were then incubated with HRP-conjugated anti-IgM (Southern Biotech) or anti-IgG (Southern Biotech) for one hour at room temperature. After another wash, the plates were developed with TMB substrate (BD Biosciences). Reactions were stopped with 1N sulfuric acid and OD450 was read using a Victor X5 plate reader (Perkin Elmer). The data was analyzed with GRAPHPAD ™ Prism and the signal was calculated as the dilution of the serum that is twice above the background. The results are shown in FIG. 24A and 24B.

As shown in this Example, the ratio of B k / A lymphocytes in both the splenic and peripheral compartments of the Vk1-39Jk5 and Vk3-20Jk1 common light chain mice demonstrated an almost wild-type pattern (Table 12 and FIG. 22 ). However, VpreBJA5 common light chain mice demonstrated fewer peripheral B lymphocytes, of which approximately 1-2% express the engineered human light chain region (data not shown). Expression levels of the Vk1-39Jk5 and Vk3-20Jk1 regions of the rearranged human light chain locus from the endogenous k light chain locus were elevated compared to an endogenous k light chain locus containing a complete replacement of segments of mouse Vk and Jk genes with human Vk and Jk gene segments (FIG. 23A, 23B, and 23C). Expression levels of the rearranged human light chain VpreBJA5 region demonstrated high expression similar to that of the endogenous k light chain locus in both heterozygous and homozygous mice (data not shown). This demonstrates that in direct competition with endogenous light chain loci A, k, or both, a single rearranged human V ^l / J ^l sequence can produce better than expression at the wild-type level from the endogenous □ light chain locus. and giving rise to the normal frequency of splenic and blood B lymphocytes. Additionally, the presence of an engineered k light chain locus having a human Vk1-39Jk5 or human Vk3-20Jk1 sequence was well tolerated by mice and appears to function wild-type by representing a substantial portion of the chain repertoire. slight in the humoral component of the immune response (FIG. 24A and 24B).

Example XIII

Reproduction of Mice that Express a Single Light Chain of the Germina1 Human Line Reordered

This example describes several other genetically modified mouse strains that can reproduce with any of the common light chain mice described herein to create multiple genetically modified mouse strains that harbor multiple genetically modified immunoglobulin loci.

Gene deletion (KO) of endogenous IgA.

To optimize the use of the engineered light chain locus, mice carrying one of the rearranged human germline light chain regions are crossed with another mouse containing a deletion at the endogenous A light chain locus. In this way, the progeny obtained will express, as their only light chain, the rearranged human germline light chain region as described in Example 11. Crossing is performed by standard art-recognized techniques and, alternatively, by a commercial breeder (eg The Jackson Laboratory). Mouse strains bearing an engineered light chain locus and a deletion of the endogenous A light chain locus are screened for the presence of the unique light chain region and the absence of endogenous mouse A light chains.

Humanized Endogenous Heavy Chain Locus.

Mice bearing an engineered human germline light chain locus are crossed with mice containing a replacement of the endogenous mouse heavy chain variable gene locus with the human heavy chain variable gene locus (see document US 6,596,541; the VELOCIMMUNE® humanized mouse, Regeneron Pharmaceuticals, Inc.). The humanized VELOCIMMUNE® mouse comprises a genome comprising human heavy chain variable regions operably linked to endogenous mouse constant region loci, such that the mouse produces antibodies comprising a human heavy chain variable region and a heavy chain constant region mouse response to antigenic stimulation. DNA encoding variable regions of antibody heavy chains is isolated and operably linked to DNA encoding human heavy chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human heavy chain of the antibody.

Mice are obtained carrying a replacement locus V ^h endogenous mouse locus with the human V ^h and V ^l a unique region of human germline reformatted endogenous locus k light chain. Reverse chimeric antibodies containing somatically mutated heavy chains (human V ^h and mouse C ^h ) with a single human light chain (human V ^l and mouse C ^l ) are obtained after immunization with an antigen of interest. Nucleotide sequences of V ^h and V ^l of B lymphocytes expressing antibodies are identified and antibodies are produced fully human by fusion of the V ^h and V ^l nucleotide sequences to human C ^h and C ^l nucleotide sequences in a suitable expression system.

Example XIV

Generation of Antibodies from Mice Expressing Human Heavy Chains and a Rearranged Human Germline Light Chain Region

After crossing mice containing the engineered human light chain region with various desired strains containing modifications and deletions of other endogenous Ig loci (as described in Example 12), the selected mice can be immunized with an antigen of interest .

Generally, a humanized VELOCIMMUNE® mouse containing one of the rearranged human germline light chain regions is exposed with an antigen, and lymphatic cells (such as B lymphocytes) are recovered from the serum of the animals. Lymphatic cells are fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and screened to identify hybridoma cell lines that produce antibodies containing variable regions of human heavy chain and light chains of rearranged human germ line that are specific to the antigen used for immunization. DNA encoding the heavy chain and light chain variable regions are isolated and bound to desirable isotypic constant regions of the heavy chain and light chain. Due to the presence of endogenous mouse sequences and any additional cis- acting elements present at the endogenous locus, the single light chain of each antibody may be somatically mutated. This adds additional diversity to the antigen-specific repertoire comprising a single light chain and various heavy chain sequences. The resulting cloned antibody sequences are subsequently expressed in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains are directly identified from antigen-specific lymphocytes.

Initially, the high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As described above, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody containing a somatically mutated human heavy chain and a single light chain derived from a rearranged human germline light chain region of the invention. Suitable human constant regions include, for example, wild-type or modified IgG1 or IgG4.

Separate cohorts of humanized VELOCIMMUNE® mice containing a replacement of the endogenous mouse heavy chain locus were immunized with human V ^h , D ^h , and J ^h gene segments and a replacement of the endogenous mouse k light chain locus with the region Engineered human germ line light chain Vk1-39Jk5 or with the engineered human germ line light chain Vk3-20Jk1 region (described above) with a human cell surface receptor protein (Antigen E). E antigen is administered directly into the back paw pad of the mice with six consecutive injections every 3-4 days. Two to three micrograms of Antigen E are mixed with 10 µg of CpG oligonucleotide (Cat. Number oligonucleotide tlrl-modn-ODN1826; InVivogen, San Diego, CA) and 25 jg of Adju-Phos (phosphate gel adjuvant from aluminum, Cat. number H-71639 -250; Brenntag Biosector, Frederikssund, Denmark) before injection. A total of six injections are administered before recall with final antigen, which is administered 3-5 days before slaughter. Bleeds after the fourth and sixth injection are collected and the immune response of the antibody is monitored by a standard antigen-specific immunoassay.

When a desired immune response was achieved, the splenocytes were collected and fused with mouse myeloma cells to preserve their viability and to form hybridoma cell lines. Hybridoma cell lines are screened and screened to identify cell lines that produce Antigen E-specific common light chain antibodies. Using this technique, several antigen E-specific common light chain antibodies (i.e., antibodies possessing variable domains) are obtained. human heavy chain, the same human light chain variable domain and mouse constant domains).

Alternatively, common light chain anti-E antigen antibodies are isolated directly from the non-fusion antigen-positive B lymphocytes to the myeloma cells, as described in U.S. 2007 / 0280945A1. Using this method, several fully human anti E Antigen common light chain antibodies (i.e., antibodies possessing human heavy chain variable domains, either an engineered human light chain Vk1-39Jk5 region or a Vk3- region) were obtained. 20Jk1 engineered human light chain, and human constant domains).

The biological properties of the examples of anti E Antigen common light chain antibodies generated according to the methods of this Example are described in detail below.

Example XV

Use of Heavy Chain Gene Segments in Antigen Specific Common Light Chain Antibodies

To analyze the structure of the human anti-E antigen light chain antibodies produced, nucleic acids encoding variable regions of heavy chain antibodies were cloned and sequenced. From the nucleic acid sequences and predicted amino acid sequences of the antibodies, the use of genes for the selected heavy chain variable region (HCVR) of selected common light chain antibodies obtained from mice was identified. Immunized humanized VELOCIMMUNE® containing the engineered human light chain Vk1-39Jk5 region or the engineered human light chain Vk3-20Jk1 region. The results are shown in Tables 14 and 15, which demonstrate that mice according to the invention generate antigen-specific common light chain antibodies from a variety of human heavy chain gene segments, due to a variety of rearrangements, when they use a mouse that expresses a light chain only from a light chain derived from human Vk1-39 or Vk3-20. The human V ^h gene segments of families 2, 3, 4, and 5 were rearranged with a variety of human D ^h segments and human J ^h segments to produce antigen-specific antibodies.

Table 14

(continuation)

Table 15

Example XVI

Determination of the Blocking Capacity of Antigen Specific Common Light Chain Antibodies Using the LUMINEX ™ Assay

Ninety-eight human common light chain antibodies raised against Antigen E were analyzed for their ability to block binding of the natural ligand of Antigen E (Ligand Y) to Antigen E in a bead-based assay.

The extracellular domain (ECD) of the E antigen was conjugated with two myc epitope tags and one 6X histidine tag (E-mmH antigen) and amine-coupled to carboxylated microspheres at a concentration of 20 | jg / ml in MONTH buffer. The mixture was incubated for two hours at room temperature followed by deactivation of the beads with 1M Tris pH 8.0 followed by washing in PBS with 0.05% (v / v) Tween-20. The beads were then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w / v) BSA (Sigma-Aldrich Corp., St. Louis, MO). In a 96-well filter plate, supernatants containing Antigen E-specific common light chain antibodies were diluted 1:15 in buffer. A negative control was prepared containing a simulated supernatant with the same media components as for the antibody supernatant. Antigen E-labeled beads were added to the supernatants and incubated overnight at 4 ° C. Biotinylated Y ligand protein was added to a final concentration of 0.06 nM and incubated for two hours at room temperature. Detection of biotinylated Y ligand bound to E-myc-myc-6HIS antigen-labeled beads was determined with Streptavidin-conjugated R-phycoerythrin (Moss Inc, Pasadena, MD) followed by measurement on a LUMINEX ™ flow cytometric-based analyzer. Background Mean Fluorescence Intensity (MFI) was subtracted from one sample without Ligand Y from all samples. The blocking percentage was calculated by dividing the MFI subtracted from the bottom of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value of 100.

In a similar experiment, the same 98 human common light chain antibodies produced against Antigen E were analyzed for their ability to block the binding of Antigen E to Y-ligand labeled beads.

Briefly, ligand Y was amine-coupled to carboxylated microspheres at a concentration of 20 µg / ml diluted in MES buffer. The mixture was incubated for two hours at room temperature followed by deactivation of the beads with 1M Tris pH 8 and then washed in PBS with 0.05% Tween-20 (v / v). The beads were then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w / v) BSA (Sigma-Aldrich Corp., St. Louis, MO). Supernatants containing Antigen E-specific common light chain antibodies were diluted 1:15 in buffer, in a 96-well filter plate. A negative control was prepared containing a simulated supernatant with the same media components as for the antibody supernatant. A biotinylated E-mmH antigen was added to a final concentration of 0.42 nM and incubated overnight at 4 ° C. The Ligand Y-labeled beads were then added to the antibody / Antigen E mixture and incubated for two hours at room temperature. Detection of bead-bound biotinylated E-mmH antigen with Ligand Y was determined with Streptavidin-conjugated R-phycoerythrin (Moss Inc, Pasadena, MD) followed by measurement on a LUMINEX ™ flow cytometric-based analyzer. Background Mean Fluorescence Intensity (MFI) was subtracted from a sample without E Antigen for example from all samples. The blocking percentage was calculated by dividing the MFI subtracted from the bottom of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.

Tables 16 and 17 show the blocking percentage for the 98 antigen E common light chain antibodies tested in both LUMINEX ™ assays. NA: not determined under current experimental conditions.

Table 16

(continuation)

(continuation)

Table 17

In the first LUMINEX ™ experiment described above, 80 common light chain antibodies containing the engineered Vk1-39Jk5 light chain were tested for their ability to block Ligand Y binding to E Antigen-labeled beads. Of these 80 Common light chain antibodies, 68 demonstrated> 50% block, while 12 demonstrated <50% block (6 25-50% block and 6 <25% block). For the 18 common light chain antibodies containing the engineered Vk3-20Jk1 light chain, 12 demonstrated> 50% block, while 6 demonstrated <50% block (3 25-50% block and 3 < 25% Blocking) of Ligand Y binding to E Antigen-labeled beads.

In the second LUMINEX ™ experiment described above, the same 80 common light chain antibodies containing the engineered Vk1-39Jk5 light chain were tested for their ability to block binding of Antigen E to Ligand Y-labeled beads. Of these 80 common light chain antibodies, 36 demonstrated> 50% block, while 44 demonstrated <50% block (27 25-50% block and 17 <25% block). For the 18 common light chain antibodies that contain the engineered Vk3-20Jk1 light chain, 1 demonstrated> 50% block, whereas 17 demonstrated <50% block (5 of 25-50% block and 12 of < 25% blocking) of Antigen E binding to Ligand Y-labeled beads.

The data in Tables 16 and 17 establish that the rearrangements described in Tables 14 and 15 generated Antigen E-specific common light chain antibodies that blocked the binding of Ligand Y to its cognate Antigen E receptor with varying degrees of efficacy, which is consistent with common light chain antibodies antigen E from Tables 14 and 15 comprising antibodies with overlapping and non-overlapping epitope specificity to Antigen E.

Example XVII

Determination of the Blocking Capacity of Antigen Specific Common Light Chain Antibodies Using ELISA

Common human light chain antibodies raised against Antigen E were analyzed for their ability to block Antigen E binding to a Ligand Y coated surface in an ELISA assay.

Ligand Y was coated onto 96-well plates at a concentration of 2 pg / ml diluted in PBS and incubated overnight followed by washing four times in PBS with 0.05% Tween-20. The plate was then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 0.5% (w / v) BSA (Sigma-Aldrich Corp., St. Louis, MO) for one hour at room temperature. In a separate plate, supernatants containing anti-E Antigen common light chain antibodies were diluted 1:10 in buffer. A sham supernatant with the same antibody components was used as a negative control. The E-mmH antigen (described above) was added to a final concentration of 0.150 nM and incubated for one hour at room temperature. The E-mmH Antigen / Antigen mixture was then added to the plate containing Ligand Y and incubated for one hour at room temperature. Detection of the E-mmH antigen bound to Ligand Y was determined with Horseradish Peroxidase (HRP) conjugated with the anti-Penta-His antibody (Qiagen, Valencia, CA) and was developed by conventional colorimetric response using tetramethylbenzidine substrate (TMB ) (BD Biosciences, San José, CA) neutralized by sulfuric acid. The absorbance was read at DO450 for 0.1 seconds. Background absorbance of a sample without E Antigen was subtracted from all samples. The blocking percentage was calculated by dividing the MFI subtracted from the bottom of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.

Tables 18 and 19 show the blocking percentage for the 98 anti E Antigen common light chain antibodies tested in the ELISA assay. NA: not determined under current experimental conditions.

Table 18

(continuation)

Table 19

As described in this Example, of the 80 common light chain antibodies containing the engineered Vk1-39Jk5 light chain tested to determine its ability to block E antigen binding to a Y Ligand coated surface, 22 demonstrated> 50 % block, while 58 demonstrated <50% block (20 25-50% block and 38 <25% block). For the 18 common light chain antibodies containing the engineered Vk3-20Jk1 light chain, one demonstrated> 50% blockade, whereas 17 demonstrated <50% blockade (5 of 25-50% blockade and 12 of < 25% blocking) of Antigen E binding to Ligand Y coated surface.

These results are also consistent with the Antigen E-specific set of common light chain antibodies comprising antibodies with overlapping and non-overlapping epitope specificity to Antigen E.

Example XVIII

Affinity Determination with BIACORE ™ for Antigen Specific Common Light Chain Antibodies

Equilibrium dissociation constants (K ^d ) for selected antibody supernatants were determined by SPR (Surface Plasmon Resonance) using a BIACORE ™ T100 instrument (GE Healthcare). All data were obtained using HBS-EP (10mM HEPES, 150mM NaCl, 0.3mM EDTA, 0.05% P20 surfactant, pH 7.4) as both sample and run buffers at 25 ° C. . Antibodies were captured from crude supernatant samples on a previously derivatized CM5 sensor chip surface with a high density of anti-human Fc antibodies using natural amine coupling chemistry. During the capture step, supernatants were injected across the surface of human anti-Fc at a flow rate of 3 pl / min, for a total of 3 minutes. The capture step was followed by an injection of running buffer or analyte at a concentration of 100 nM for 2 minutes at a flow rate of 35 pl / min. The dissociation of the antigen from the captured antibody was monitored for 6 minutes. The captured antibody was removed by brief injection of 10 mM glycine, pH 1.5. All sensorgrams were referenced twice by subtracting the sensorgrams of the buffer injections from the analyte sensorgrams, thus removing artifacts caused by dissociation of the antibody from the capture surface. Binding data for each antibody was fitted to a 1: 1 binding model with mass transport using the BIACORE ™ T100 v2.1 evaluation software. The results are shown in Tables 20 and 21.

Table 20

(continuation)

Table 21

The binding affinities of common light chain antibodies comprising the rearrangements shown in Tables 14 and 15 vary, and almost all show a K ^d in the nanomolar range. The affinity data is consistent with the common light chain antibodies that are the result of the combinatorial association of rearranged variable domains described in Tables 14 and 15 that are high affinity, clonally selected and somatically mutated. Along with the data shown above, the common light chain antibodies described in Tables 14 and 15 comprise a collection of diverse high affinity antibodies that show specificity for one or more epitopes on the E Antigen.

Example XIX

Determination of the Binding Specificities of Antigen Specific Common Light Chain Antibodies Using the LUMINEX ™ Assay

The ability of selected antigen E common light chain antibodies to bind to the ECD of the ECD variants of Antigen E and Antigen E was evaluated, including the ortholog of mono cynomolgus (Antigen E Mf), which differs from human protein. in approximately 10% of its amino acid residues; an E Antigen deletion mutant lacking the last 10 amino acids of the C-terminus of the ECD (E-ACT Antigen); and two mutants containing an alanine substitution at sites suspected of interaction with Ligand Y (E-Ala1 Antigen and E-Ala2 Antigen). The Antigen E proteins were produced in CHO cells and each contained a C-terminal myc-myc-His tag.

For binding studies, the Antigen E ECD protein or variant protein (described above) from 1 ml of culture medium was captured by incubation for 2 hours at room temperature with 1 x 10 6 microspheres (Luminex ™) coated covalent with an anti-myc monoclonal antibody (MAb 9E10, CRL-1729 ™ hybridoma cell line; ATCC, Manassas, VA). The beads were washed with PBS before use. Supernatants containing common antigen E light chain antibodies were diluted 1: 4 in buffer and added to 96-well filter plates. A mock supernatant without antibodies was used as a negative control. Beads containing the captured Antigen E proteins were then added to the antibody samples (3,000 beads per well) and incubated overnight at 4 ° C. The next day, the sample beads were washed and the bound common light chain antibody was detected with an anti-human IgG antibody conjugated to R-phycoerythrin. The fluorescence intensity of the beads (approximately 100 beads counted for each antibody sample that binds each Antigen E protein) was measured with a Luminex ™ flow cytometric-based analyzer, and recorded the median fluorescence intensity (MFI) for at least 100 beads counted per bead / antibody interaction. The results are shown in Tables 22 and 23.

Table 22

(continuation)

Table 23

Claims (7)

1. A mouse whose genome comprises: (a) a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human Vh segment, at least one non-rearranged human Dh, and at least one non-rearranged human Jh operably linked to a heavy chain constant region gene endogenous; (b) a humanized immunoglobulin light chain variable locus comprising not more than one, or not more than two, rearranged human light chain V / J sequences operably linked to an endogenous light chain constant region gene; and, (c) an ectopic nucleic acid sequence expressing a functional mouse ADAM6a protein or a functional ortholog or a functional homolog or a functional fragment thereof and an ectopic nucleic acid sequence expressing a functional mouse ADAM6b protein or an ortholog functional or a functional homolog or a functional fragment thereof, the ADAM6 proteins, orthologs, homologs or fragments being functional in a male mouse, wherein said function is associated with male fertility, said mouse having the function of eliminated or reduced endogenous ADAM6 and further characterized in that its germline lacks a non-rearranged endogenous immunoglobulin gene segment Vk and a non-rearranged endogenous immunoglobulin gene segment Jk. 2. The mouse according to claim 1, wherein the ectopic nucleic acid sequences are at a locus other than the immunoglobulin heavy chain variable locus. 3. A mouse whose genome comprises: a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human Vh segment, at least one non-rearranged human DH, and at least one non-rearranged human JH operably linked to an endogenous heavy chain constant region gene; a humanized immunoglobulin light chain variable locus comprising a single rearranged human light chain V / J sequence operably linked to an endogenous light chain constant region gene, wherein the mouse expresses a single chimeric light chain, and wherein the mouse expresses from ectopic nucleic acid sequences a functional mouse ADAM6a protein and a functional mouse ADAM6b protein, whose ADAM6a and ADAM6b proteins are functional in a male mouse, where said function is associated with male fertility, said mouse having the function of eliminated or reduced endogenous ADAM6 and further characterized in that its germline lacks a non-rearranged endogenous immunoglobulin gene segment Vk and a non-rearranged endogenous immunoglobulin gene segment Jk. 4. The mouse of claim 3, wherein the ectopic nucleic acid sequences are in a position that is not within an endogenous immunoglobulin heavy chain locus. 5. A mouse cell whose genome comprises: a humanized immunoglobulin heavy chain variable locus comprising at least one non-rearranged human Vh segment, at least one non-rearranged human DH, and at least one non-rearranged human JH operably linked to an endogenous heavy chain constant region gene; a humanized immunoglobulin light chain variable locus comprising not more than one, or not more than two, rearranged human light chain V / J sequences operably linked to an endogenous light chain constant region gene; and, an ectopic nucleic acid sequence expressing a functional mouse ADAM6a protein or a functional ortholog or a functional homolog or a functional fragment thereof and an ectopic nucleic acid sequence expressing a functional mouse ADAM6b protein or a functional ortholog or a functional homolog or a functional fragment thereof, ADAM6 proteins, orthologs, homologs or fragments being functional in a male mouse, where said function is associated with male fertility, said cell having the function of eliminated or reduced endogenous ADAM6 and further characterized in that it lacks a non-rearranged endogenous immunoglobulin Vk gene segment and a non-rearranged endogenous immunoglobulin Jk gene segment. 6. The mouse cell of claim 5, wherein the mouse cell comprises a rearranged human light chain V / J gene sequence. 7. The mouse cell of any one of claims 5 or 6, wherein the cell is an embryonic stem cell (ES).